Novel therapeutic target for protozoal diseases

ABSTRACT

A novel Hemozoin Detoxification Protein (HDP) from  Plasmodium  and related parasites is provided as a target for therapeutic intervention in diseases caused by the parasites. HDP has been shown to play a critical role in adhesion to, or invasion into, host cells by the parasite. Furthermore, HDP catalyzes the neutralization of heme by the parasite, by promoting its polymerization into hemozoin. This invention provides methods and compositions for therapies based on the administration of protein, DNA or cell-based vaccines and/or antibodies based on HDP, or antigenic epitopes of HDP, either alone or in combination with other parasite antigens. Methods for the development and use of compounds that inhibit the catalytic activity of HDP, and diagnostic and laboratory methods utilizing HDP are also provided. HDP is also referred to herein as Fasciclin Related Adhesive Protein (FRAP).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of and claims benefit of U.S.patent application Ser. No. 11/249,355, the complete contents of whichare hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to therapies for the treatment andprevention of certain parasitic diseases. In particular, the inventionprovides method of inhibiting the ability of Heme Detoxification Protein(HDP) to form hemozoin from heme, thereby treating or preventingdiseases caused by Plasmodium and/or Theileria species.

2. Background of the Invention

Malaria, a blood-borne infection caused by Plasmodium parasites, is amajor health issue in the tropics, with 300-500 million clinicalepisodes of this disease occurring each year. A licensed vaccine againstmalaria is not available and the parasite is developing resistanceagainst most of the currently available antimalarials. There is anurgent need to develop new therapeutics (drugs and vaccines) againstmalaria, which will reduce the morbidity and mortality associated withthis disease. The genome of Plasmodium falciparum has been sequenced andcan be exploited to understand the molecular basis of the onset andsustenance of infection by these pathogens. Deciphering these mechanismswill unravel the complex interplay between the troika of host, pathogenand its environment, which is vital for identifying new targets forintervention.

Malaria infection starts with the introduction of Plasmodium sporozoitesinto the blood stream of its human host, when it is bitten by aninfected mosquito. Of the four Plasmodium species that infect humans, P.falciparum is the most virulent—resulting in severe anemia and cerebralmalaria, which can be fatal. Fewer than 200 sporozoites are introducedand even fewer succeed in invading liver cells, the target organ for theonset of malaria infection in a host. A successful adhesion and livercell invasion by the sporozoite is critical for this onset and istherefore, the Achilles heel of the parasite. Once inside the livercell, the parasite rapidly multiplies and within a few days releasesthousands of parasites, which leads to the clinical pathology of thisdisease. Therefore, an ideal approach to control malaria is to develop avaccine or therapeutic, which either prevents the sporozoite frominfecting liver cells or destroys the parasite during liver stages ofits life cycle. Such a vaccine is feasible as animals and humanvolunteers immunized with Plasmodium sporozoites that have beenattenuated by exposure to X-Ray or gamma radiation, are protected whensubsequently challenged with infectious sporozoites (Hoffman, et al.(2002) J Infect Dis, 1155-1164; Nussenzweig et al. (1967) Protectiveimmunity produced by the injection of x-irradiated sporozoites ofPlasmodium berghei. Nature, 216, 160-162.). While this groundbreakingdiscovery clearly indicated that it is feasible to make a vaccineagainst malaria, the biggest stumbling block for malaria researchersworldwide has been to decipher the parasite antigens recognized by thehost and to understand the immune mechanisms underlying this protection.Extensive immunological studies with known sporozoite antigens haveconcluded that this protection is not conferred due to a dominant immuneresponse against a single antigen but is mediated by the summation ofmany modest humoral and cell-mediated immune responses against a largevariety of antigens, many of which are currently not known (Hoffman, S.(1996) Malaria Vaccine Development: A multi immune response approach.ASM press, Washington, D.C.). Identification of these antigens is notonly the major challenge, it is vital for the development of asuccessful vaccine against malaria.

Historically, antigen(s) selected as a vaccine candidate in a givenpathosystem are (i) present on the surface of the pathogen, (ii) aregenerally involved in host-pathogen interactions and are therefore, oneof the first molecules that are recognized by the host immune system(Moxon, R. and Rappuoli, R. (2002) Br Med Bull, 62, 45-58). Thesecriteria are also valid for malaria parasite as the two major vaccinecandidates viz., Circumsporozoite protein (CSP) (Cerami, C. et al.(1992) Cell, 70, 1021-1033) and Thrombospondin-related anonymous protein(TRAP) (Robson, et al. (1995) Embo J, 14, 3883-3894) are involved in theinvasion of liver cells by the parasite.

Upon entering red blood cells, the Plasmodium parasite undergoes rapidmultiplication giving rise to 28-32 parasites in less than 48 hours.Hemoglobin represents ˜95% of the total RBC content, and the parasitedigests up to 75% of the hemoglobin, which serves as its source of aminoacids. While this process of hemoglobin digestion provides the parasitewith a ready source of amino acids, it also releases free heme, which inthe absence of a globin moiety, is extremely toxic for the parasite(Gluzman, et al. (1994) J Clin Invest, 93, 1602-1608.). The parasitesurvives by effectively neutralizing toxic heme into a non-toxic andpolymerized product known as hemozoin, which is chemically identical toβ-hematin (Francis,et al. (1997) Annu Rev Microbiol, 51, 97-123. Most ofthe currently available antimalarials have been shown to be binding tofree heme, which inhibits its polymerization, and the toxicity resultingfrom the free heme causes the death of the parasite (Slater and Cerami(1992) Nature, 355, 167-169). Therefore, pathway(s) that lead tohemozoin formation are extremely attractive drug targets. Unfortunately,the mechanism(s) in use by the parasite for the polymerization processis poorly understood. Two parasite proteins viz., Histidine rich proteinII and III have been proposed to be responsible for this activity(Sullivan, et al. (1996) Science, 271, 219-222.), though parasiteslacking either or both of the proteins make copious amounts of hemozoinwithout any loss of activity (Wellems, et al. (1991) Proc Natl Acad SciUSA, 88, 3382-3386). Therefore, an unknown protein(s) has been longthought to be responsible for this activity.

The prior art has thus far failed to provide satisfactory vaccines ordrug therapies to combat diseases caused by parasites such asPlasmodium. There is thus an ongoing need to identify and characterizepotential targets for such therapeutic intervention.

SUMMARY OF THE INVENTION

The parasite protein “Fasciclin Related Adhesive Protein” (“FRAP”),which is also referred to as “Heme Detoxification Protein (“HDP”)herein, has been discovered, and its use as a target for therapeuticintervention in parasitic diseases is described herein. The designations“FRAP” and “HDP” are used interchangeably herein. FRAP (HDP) isexpressed during the infective forms of parasites such as Plasmodium andTheileria, is intimately involved in the onset of parasitic infections,and key sequences of the protein are highly conserved across Plasmodiumspecies and related genera. Thus, this protein is an ideal target forthe treatment and/or prevention of parasitic diseases by a variety ofmethods, including vaccine development. In addition, FRAP (HDP)catalyzes the neutralization of toxic heme into non-toxic hemozoin.Thus, FRAP (HDP) is an attractive target for inhibitory drug therapies.Such therapies may include, for example, the use of compounds that bindto the HDP protein to either prevent the binding of heme, or to preventthe conversion of bound heme to hemozoin. Alternatively, such therapiesmay involve the use of compounds that bind to heme to prevent it frombinding to HDP, or to prevent its conversion to hemozoin after binding.The details of these and other mechanisms of action are described indetail below.

The present invention provides a composition for eliciting an immuneresponse to Plasmodium. The composition comprises a substantiallypurified synthesized or recombinant protein comprising an amino acidsequence represented by SEQ ID NO: 1 or SEQ ID NO: 25; or asubstantially purified synthesized or recombinant protein comprising anamino acid sequence that displays at least 90% identity to SEQ ID NO: 1or SEQ ID NO: 25. The composition may further include at least one of:one or more additional antigens, and one or more adjuvants. Thecomposition may further include one or more additional peptides,polypeptides or proteins each of which is different from saidsubstantially purified synthesized or recombinant protein.

The invention also provides a composition for eliciting an immuneresponse to Plasmodium, which comprises a substantially purifiedsynthesized or recombinant peptide, polypeptide or protein comprising anamino acid sequence represented by SEQ ID NO: 37. The substantiallypurified synthesized or recombinant peptide, polypeptide or protein maycomprise an amino acid sequence represented by SEQ ID NO: 24, or anamino acid sequence that displays at least about 85% identity to SEQ IDNO: 24. The composition may further include at least one of: one or moreadditional antigens, and one or more adjuvants. The composition mayfurther include one or more additional peptides, polypeptides orproteins each of which is different from the substantially purifiedsynthesized or recombinant peptide, polypeptide or protein.

In addition, the invention provides a vaccine comprising a substantiallypurified synthesized or recombinant protein comprising an amino acidsequence represented by SEQ ID NO: 1 or SEQ ID NO: 25; or asubstantially purified synthesized or recombinant protein comprising anamino acid sequence that displays at least 90% identity to SEQ ID NO: 1or SEQ ID NO: 25. The vaccine may further include at least one of one ormore additional antigens, and one or more adjuvants.

In another embodiment, the invention provides a vaccine comprising asubstantially purified synthesized or recombinant peptide, polypeptideor protein comprising an amino acid sequence represented by SEQ ID NO:37. The substantially purified synthesized or recombinant peptide,polypeptide or protein may comprise an amino acid sequence representedby SEQ ID NO: 24, or an amino acid sequence that is at least 85%identical to SEQ ID NO: 24. The vaccine may include at least one of: oneor more additional antigens, and one or more adjuvants. The vaccine mayfurther include one or more additional peptides, polypeptides orproteins each of which is different from the substantially purifiedsynthesized or recombinant peptide, polypeptide or protein.

In another embodiment, the invention provides a substantially purifiedsynthesized or recombinantly produced antibody specific for: a proteinwith an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO:25; or a protein with an amino acid sequence that displays at least 90%identity to SEQ ID NO: 1 or SEQ ID NO: 25. In some embodiments, theantibody is chimeric, humanized, or fully human.

In another embodiment, the invention provides a substantially purifiedsynthesized or recombinantly produced antibody specific for: a peptidewith an amino acid sequence represented by SEQ ID NO: 37, or a peptidewith an amino acid sequence represented by SEQ ID NO: 24. In someembodiments, the antibody is chimeric, humanized, or fully human.

The invention further provides a transfected cell comprising expressablerecombinant DNA that encodes: one or more of a peptide, polypeptide orprotein which is or includes an amino acid sequence represented by SEQID NO: 1, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 37; or one or moreof a peptide, polypeptide or protein which is or includes an amino acidsequences that displays at least 90% identity with one or more of SEQ IDNO: 1, SEQ ID NO: 25, or SEQ ID NO: 37, or at least about 85% identitywith SEQ ID NO: 24. In another embodiment, such transfected cells areused to elicit an immune response and/or to serve as a vaccine.

In yet another embodiment, the invention provides a method of treatingor preventing a disease caused by a Plasmodium parasite in a patient inneed thereof. The method comprises the step of administering to thepatient one or more antibodies specific for one or more amino acidsequences represented by SEQ ID NO: 1, SEQ ID NO: 24, SEQ ID NO: 25 orSEQ ID NO: 37.

The antibody may be synthesized or recombinantly produced.

In yet another embodiment, the invention provides a method of elicitingan immune response to a Plasmodium parasite in a patient in needthereof. The method comprises the step of administering to the patientone or more peptides, polypeptides or proteins which comprise one ormore amino acid sequences selected from the group consisting of SEQ IDNO: 1, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 37, and amino acidsequences which display at least 90% identity with SEQ ID NO: 1, SEQ IDNO: 25, SEQ ID NO: 37, or at least about 85% identity with SEQ ID NO:24. The peptides, polypeptides or proteins may be synthesized orrecombinantly produced.

In yet another embodiment, the invention provides a method of treatingor preventing a disease caused by a Plasmodium or Theileria parasite ina patient in need thereof. The method comprises the step ofadministering to the patient a compound that inhibits FRAP protein. Inone embodiment, the patient is an animal. In one embodiment, thecompound is an antibody.

In some instances, the compound interacts with a peptide, polypeptideprotein that comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 19,SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 37. In addition, thecompound may bind to or interact with one or more of amino acid residuesF42, H44 and H122 of FRAP protein encoded by SEQ ID NOS: 1, 7 and 11, orwith one or more equivalent amino acid residues in other FRAP proteins,i.e. amino acid residues that fulfill the same or a similar function inanother FRAP protein, such as the proteins encoded by SEQ ID NO: 3, SEQID NO: 5, or SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17and SEQ ID NO: 19.

In yet another embodiment, the invention provides a whole organismvaccine against a parasite. The vaccine comprises an attenuated parasitewhich is unable to produce a fully functional FRAP protein. Theattenuated parasite may include one or more mutations or deletions in acoding region that encodes the fully functional FRAP protein. One ormore mutations may be in a coding region that encodes the fullyfunctional FRAP protein at a site which encodes for an amino acidresidue selected from the group consisting of phenylalanine 42,histidine 44, phenylalanine 64, histidine 79, phenylalanine 90,histidine 122, cysteine 191, histidine 192 and histidine 197 of FRAPproteins encoded by SEQ ID NOS: 1, 7 and 11, or the equivalent aminoacid residues in other FRAP proteins, i.e. amino acid residues thatfulfill the same or a similar function in another FRAP protein, such asthe proteins encoded by SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 9, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19. In oneembodiment, the parasite is unable to produce a fully functional FRAPprotein due to RNA silencing. In another embodiment, the parasite isunable to produce normal levels of a fully functional FRAP protein dueto attenuation of a promoter that is operably linked to DNA encodingFRAP.

The invention also provides a method for high throughput screening forantimalarial agents that inhibit the conversion of heme to hemozoin. Themethod comprises the steps of: providing a potential antimalarial agent;determining a first level of conversion of heme substrate to hemozoin byFRAP in the presence of said potential antimalarial agent, and a secondlevel of conversion of heme substrate to hemozoin by FRAP in the absenceof said potential antimalarial agent; and comparing said first level ofconversion to said second level of conversion, wherein if said secondlevel of said conversion is higher than said first level of conversion,said potential antimalarial agent inhibits the conversion of heme tohemozoin. In some embodiments, FRAP has one or more amino acid sequencesselected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 15, SEQ ID NO: 17, and SEQ ID NO: 19.

The invention also provides a method for expression and purification ofa recombinant protein. The method comprises the step of providing avector that operably encodes the recombinant protein, wherein saidrecombinant protein comprises one or more of SEQ ID NO: 1 or SEQ ID NO:25. The recombinant protein may be a fusion protein, and may compriseone or more copies of SEQ ID NO: 24 or SEQ ID NO: 37. The vector mayalso encode an antigen such as CSP or TRAP.

The invention also provides a method for diagnosing prior exposure toPlasmodium or Theileria. The method comprises the steps of: obtaining abiological sample from a patient and determining whether at least one ofan amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO:25 and SEQ ID NO: 37, or an antibody to at least one of SEQ ID NO: 1,SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25and SEQ ID NO: 37 is present in said biological sample.

The invention also provides a diagnostic assay for determining exposureto Plasmodium or Theileria, comprising: one or more substances capableof selectively binding i) at least one amino acid sequence selected fromthe group consisting of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17, SEQID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 37; or ii) anantibody to at least one of SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 37; and oneor more labels which are activated upon binding by said one or moresubstances.

The invention also provides a method for identifying compounds thatinhibit heme neutralization by FRAP. The method comprises the steps ofa) contacting FRAP, or an extract containing FRAP, with a known amountof heme, in the presence or absence of a known dilution of a testcompound; and b) quantitating a percent inhibition of said hemeneutralization by said test compound by comparing differences in saidheme neutralization in the presence and absence of said test compound.FRAP may have one or more amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO:17, and SEQ ID NO: 19.

The invention also provides a method for diagnosing exposure (prior orongoing) to Plasmodium or Theileria. The method comprises the steps of:obtaining a biological sample from a patient and determining whether atleast one of a nucleic acid sequence selected from the group consistingof SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO:25, SEQ ID NO: 26 and SEQ ID NO: 38, is present in said biologicalsample. The step of determining may be performed using polymerase chainreaction.

The invention also provides a diagnostic kit or assay for determiningexposure (prior or ongoing) to Plasmodium or Theileria. The kit or assaycomprises: one or more nucleic acids which hybridize to one or morenucleic acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 8, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO:38 and SEQ ID NO: 39; and a mechanism for detecting hybridization. Thekit may further comprise means for quantifying an amount ofhybridization, and the one or more nucleic acids may be bound to asubstrate, such as a biochip.

The invention further provides a composition for eliciting an immuneresponse to Plasmodium. The composition comprises a nucleic acidsequence encoding an amino acid sequence represented by SEQ ID NO: 1.SEQ ID NO: 7 or SEQ ID NO: 25. The nucleic acid sequence may be SEQ IDNO: 2, SEQ ID NO: 8, or SEQ ID NO: 26 or a sequence that displays atleast 90% homology to SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 26. Thecomposition may contain one or more adjuvants. The composition maycontain a nucleic acid encoding one or more peptides, polypeptides orproteins which are not encoded by SEQ ID NO: 2, SEQ ID NO: 8, or SEQ IDNO: 26. In one embodiment, the nucleic acid sequence is contained in avector, for example, an adenoviral vector.

The invention also provides a composition for eliciting an immuneresponse to Plasmodium which comprises a nucleic acid sequence encodingthe amino acid sequence represented by SEQ ID NO: 37. In one embodiment,the nucleic acid sequence comprises a nucleic acid sequence encoding anamino acid sequence represented by SEQ ID NO: 24. The nucleic acidsequence may be SEQ ID NO: 38 or SEQ ID NO: 39, or a sequence thatdisplays at least 90% homology to SEQ ID NO: 38, or a sequence thatdisplays at least 85% homology to SEQ ID NO: 39. The composition maycontain one or more adjuvants, and may further comprise nucleic acidsencoding one or more peptides, polypeptides or proteins which are notencoded by SEQ ID NO: 38 or SEQ ID NO: 39. In one embodiment, thenucleic acid sequence is contained in a vector, for example, anadenoviral vector.

The invention also provides a vaccine for eliciting an immune responseto Plasmodium, the vaccine comprising a nucleic acid sequence encodingan amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO: 7 or SEQID NO: 25. In some embodiments, the nucleic acid sequence is SEQ ID NO:2, SEQ ID NO: 8, or SEQ ID NO: 26, or a sequence that displays at least90% homology to SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 26. Thecomposition may contain one or more adjuvants, and may comprise anucleic acid encoding one or more peptides, polypeptides or proteinswhich are not encoded by SEQ ID NO: 2, SEQ ID NO: 8, or SEQ ID NO: 26.In one embodiment, the nucleic acid sequence is contained in a vector,for example, an adenoviral vector.

The invention further provides a vaccine for eliciting an immuneresponse to Plasmodium, the vaccine comprising a nucleic acid sequenceencoding an amino acid sequence represented by SEQ ID NO: 37. In oneembodiment, the nucleic acid sequence comprises a nucleic acid sequenceencoding an amino acid sequence represented by SEQ ID NO: 24. Thenucleic acid sequence may be SEQ ID NO: 38 or SEQ ID NO: 39, or asequence that displays at least 90% homology to SEQ ID NO: 38, or asequence that displays at least 85% homology to SEQ ID NO: 39. Thecomposition may contain one or more adjuvants, and may comprise nucleicacids encoding one or more peptides, polypeptides or proteins which arenot encoded by SEQ ID NO: 38 or SEQ ID NO: 39. In one embodiment, thenucleic acid sequence is contained in a vector, for example, anadenoviral vector.

The invention further provides a vaccine for eliciting an immuneresponse to Theileria, the vaccine comprising a nucleic acid sequenceencoding an amino acid sequence represented by SEQ ID NO: 17 or SEQ IDNO: 19. In some embodiments, the nucleic acid sequence is SEQ ID NO: 18or SEQ ID NO: 20, or a sequence that displays at least 90% homology toSEQ ID NO: 18 or SEQ ID NO: 20.

The invention further provides a method of treating or preventing adisease caused by a Plasmodium or Theileria parasite in an individual inneed thereof. The method comprises the step of inhibiting interaction ofheme and Heme Detoxification Protein (HDP) in the individual. Suchindividuals are typically mammals, and can be of any species that aresusceptible to infection by Plasmodium or Theileria parasites, e.g.humans, cows, etc.

In one embodiment of the invention, the step of inhibiting is carriedout by administering to the individual one or more compounds thatinhibit interaction of heme and HDP. In some cases, the one or morecompounds bind to heme and may, for example, 1) prevent heme frombinding to HDP, or 2) allow the binding of heme to HDP but preventdetoxification of heme by HDP. In other embodiments, the one or morecompounds bind to HDP and may, for example, 1) prevent binding of hemeto HDP, 2) prevent the production of hemozoin from bound heme, 3) bindat the active site of HDP, or 4) bind at an allosteric site of HDP. Inother embodiments, the step of inhibiting is carried out by modificationof a cell membrane of the Plasmodium or Theileria parasite. In yetanother embodiment, the step of inhibiting is carried out by inhibitingsecretion of HDP from the Plasmodium or Theileria parasite.

In a preferred embodiment of the inveniton, the disease that is treatedor prevented is malaria. In this case, the compound may be administeredto an individual in combination with one or more of: an additionalantimalarial agent, an agent for reversing antimalarial resistance, andan adjuvant. Administration of the compound may be prior to, concurrentwith, or subsequent to administration of the additional antimalarialagent or said agent for reversing antimalarial resistance. Suitableadditional antimalarial agents include a) quinolines, b) folic acidantagonists, c) sulfonamides, and d) antibiotics. Suitable agents forreversing antimalarial resistance are, for example, inhibitors ofmultidrug resistance. Administration may be accomplished, for example,orally, parenterally, sublingually, rectally, topically or with aninhalation spray.

The invention further provides a method of treating an individualinfected with Plasmodium or Theileria or who has been or will be exposedto Plasmodium or Theileria, The method comprises the step of providingthe individual with one or more compounds that inhibit the ability ofHDP to produce hemozoin from heme. In some cases, the one or morecompounds bind to heme and may, for example, 1) prevent heme frombinding to HDP, or 2) allow the binding of heme to HDP but preventdetoxification of heme by HDP. In other embodiments, the one or morecompounds bind to HDP and may, for example, 1) prevent binding of hemeto HDP, 2) prevent the production of hemozoin from bound heme, 3) bindat the active site of HDP, or 4) bind at an allosteric site of HDP.

The invention further provides a method for identifying compounds thatinhibit HDP expression. The method comprises the steps of a) contactingPlasmodium with a test compound and b) determining whether thePlasmodium expresses HDP. The step of determining may be carried out,for example, by measuring mRNA or by measuring HDP.

The invention further provides pharmaceutical compositions comprising apharmaceutically acceptable carrier and an antimalarially effectiveamount of at least one compound selected Table 11 below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-J. This figure shows the amino acid sequences of the FRAPprotein in a variety of organisms as follows: A, Plasmodium falciparum;B, Plasmodium vivax; C, Plasmodium gallinaceum; D, Plasmodium knowlesi;E, Plasmodium reichenowi; F, Plasmodium yoelii; G, Plasmodium berghei;H, Plasmodium chaubaudi; I, Theileria parva, and J, Theileria annulata.

FIG. 2A-J. This figure shows the nucleic acid sequences that encode theFRAP protein in a variety of organisms as follows: A, Plasmodiumfalciparum; B, Plasmodium vivax; C, Plasmodium gallinaceum; D,Plasmodium knowlesi; E, Plasmodium reichenowi; F, Plasmodium yoelii; G,Plasmodium berghei; H, Plasmodium chaubaudi; I, Theileria parva, and J,Theileria annulata. The sequences represent the coding sequence of FRAPfrom different parasites. The gene itself is present on three separateexons and the sequence provided below is intron-free and represents onlythe coding sequence of the protein.

FIG. 3: Multiple sequence alignment of FRAP from Plasmodium andTheileria parasites. Sequences were aligned using the Clustal Walgorithm. Amino acids in bold (60 total) represent residues that areconserved across the two genera of phylum apicomplexa. Residues markedwith an asterisk represent amino acid positions that are identical onlyin the Plasmodial genus. Overall, the Plasmodial sequences have 60%sequence identity. FAS1 domain of FRAP has been aligned with theconsensus sequence of FAS1 domain (SEQ ID NO: 21) and has an e-value of2e-10. The two conserved motifs have been underlined.

FIG. 4. Schematic representation of P. falciparum FRAP gene organizationand the expressed recombinant proteins. (A) FRAP represents the fulllength protein encoding 205 amino acids. FRAP 2 represents a truncatedversion of the full length protein containing only amino acids 1-87,while FRAP 3 represents amino acids 88-205, encoding the Fasciclin 1domain. (B) RT-PCR analysis of PfFRAP. DNA encoding the coding region ofFRAP was amplified by RT-PCR using total RNA from sporozoite stage ofthe parasite's lifecycle. The amplification was performed in thepresence (+RT) and absence of reverse transcriptase (−RT) to rule outthe direct amplification from any contaminating genomic DNA. (C)Recombinant Expression and Purification of PfFRAP proteins. Full-lengthFRAP (lane 1) and its truncated variants, FRAP2 (lane 2) and FRAP3 (lane3) were purified to homogeneity by a two step chromatography. (D)Western Blot analysis. Purified proteins were resolved on a 12% Nu-PAGEgel; transferred onto a nitrocellulose membrane and the membrane wasprobed using anti-FRAP2 antibody followed by an anti-mouse HRPconjugate.

FIG. 5. Binding analysis of FRAP proteins on HepG2 cells. Five differentconcentrations of recombinant proteins were investigated for theirpotential to bind to liver cells. Bound protein was detected usinganti-polyhistidine monoclonal followed by the addition of anti-mousealkaline phosphatase conjugate and a fluorescent substrate. Fluorescencewas measured using a plate reader with excitation at 350 nm and emissionat 460 nm. Black bars: CS protein; Hashed bars: FRAP; Grey bars: FRAP2;White bars: FRAP3.

FIG. 6. Nature of FRAP receptor on liver cells. Binding activity of theFRAP proteins was evaluated on liver cells in the absence or presence ofdifferent concentrations of heparin and Chondroitin sulfate A. Panel A:FRAP; Panel B: FRAP2. Blank and hashed bars represent inhibition ofbinding activity in the presence of different concentrations of heparinand chondroitin sulfate A, respectively. FIG. 7. Overlap betweenFRAP-based peptides. Ten overlapping peptides spanning the FRAP2sequence were synthesized and utilized for the identification ofregions(s) recognized by antibodies specific for FRAP.

FIG. 8. FRAP-mediated neutralization of toxic heme into non-toxicHemozoin. 500 pmoles of each of the protein was incubated with differentconcentrations of free heme at 37° C. for 16 hours, under acidicconditions (500 mM Sodium acetate pH 5.2). After 16 hours, free heme wasremoved by washing and the insoluble pellet representing hemozoin wassolubalized in sodium hydroxide and estimated using a spectrophotometer.FRAP showed 10-20 fold more activity in comparison to HRPII, indicatingthat it could be the major protein responsible for polymerization ofheme in the parasite.

FIG. 9. FRAP-mediated hemozoin formation requires intact protein.Hemozoin formation was investigated with FRAP pretreated with proteinaseK, a nonspecific protease or with buffer alone. Incubation of FRAP withProteinase K led to a complete loss of activity suggesting that theconversion of heme into hemozoin requires intact FRAP protein.

FIG. 10. Chemical structure of hemozoin. Dimerization of heme through aFe1-O41 linkage leads to the formation of β-hematin. Oxygen mediatednon-covalent interaction between β-hematin units leads to the stackingand the polymerized product is known as hemozoin. Adapted from (Pagolaet al., 2000)

FIG. 11. Spectroscopic verification of FRAP-mediated polymerized heme ashemozoin. Heme polymerized into hemozoin was subjected to Fouriertransform-Infra Red (FT-IR) spectroscopy to verify its chemical nature.The insoluble product showed a dramatic decrease in transmittance at1664 and 1211 cm⁻¹, a well established spectroscopic signature ofβ-hematin.

FIG. 12. Time Kinetics of hemozoin formation. FRAP-mediated hemozoinformation was investigated with respect to time. 500 pmoles of proteinwas incubated with 300 nmoles of heme for different times and the amountof heme polymerized was measured as previously described. Hemozoinformation was found to be essentially complete by 5 hours.

FIG. 13. Stoichiometry of FRAP-Heme Interaction. Stoichiometry of theFRAP-Heme interaction was determined spectrophotometrically bycontinuous variation method (Job Plot). Change in absorbance wasmeasured by using different molar ratios of FRAP-heme complex. FRAP-Hemehave a 1:1 stoichiometry.

FIG. 14. Inhibition of FRAP-mediated hemozoin formation by Chloroquine.Hemozoin formation was investigated in the absence or presence ofdifferent concentrations of chloroquine, an antimalarial drug with highaffinity for heme. Chloroquine inhibited heme polymerization in a dosedependent manner. This indicates that blocking FRAP-Heme interactioncould serve as an effective antimalarial strategy.

FIG. 15A and B. A, amino acid and B, nucleic acid encoding the FRAP2derivative of FRAP.

FIG. 16A-F. HDP detoxifies and sequesters heme as Hz. a, HDP (black bar)mediated Hz production is dose dependent and could be up to 20 foldhigher than HRP II (light grey bar), oleic acid (dark grey bar) ormono-oleoyl glycerol (white bar). Values are mean ±s.d b, Hz productionincreases, with increasing amount of HDP (0-0.5 nmol) in a reactioncontaining 300 nmol of free heme. c, Fourier transform infrared spectrumof HDP-derived product showed absorption peaks at 1660 and 1210 cm-1,which validated it as Hz. d, HDP-mediated Hz production is restricted toa pH range found inside the food vacuole. e, Native P. falciparum HDPpurified from intraerythrocytic parasites. Silver stained gel (leftpanel), Immunoblot (right panel). f, Native HDP (black bar) can produceHz. Hashed bar represents recombinant protein.

FIG. 17A-B. HDP gene is important for the survival of the parasite. a,Schematic representation of strategy used for targeting HDP locusthrough single cross over recombination. The anticipated cross-overevent at the HDP locus and restriction enzyme sites Bam HI (B) and EcoRV (E) are shown. b, Lanes a and b depict Bam HI-linearized pHDPKO (6.3kb) and Bam HI and Eco RV digested DNA from wild type P. falciparumparasites containing the HDP locus (5.3 kb), respectively. Parasitessurviving after three selection cycles (lanes c, d) had an intact HDPlocus and an episomal copy of the pHDPKO plasmid expressing hDHFR. Barrepresents 500 bp.

FIG. 18A-C. Structural and biochemical analysis of HDP-mediated Hzformation.a, Heme (100 μM) solution was titrated into protein (5 μM) andthe heat evolved was measured by Isothermal titration calorimetry.Binding isotherm integrating the data from the top panel. b, Full lengthHDP is necessary for Hz formation as HDP2 (circle) and HDP3 (triangle)alone could not produce Hz. c, Hz formation activity of P. yoelii HDP(grey bars) is indistinguishable from its P. falciparum ortholog (blackbars). Values are mean ±s.d. with data from at least two independentexperiments.

FIG. 19A-D. Cloning, expression and purification of HDP proteins. a,RT-PCR amplification of HDP coding sequence. b, Schematic representationof HDP gene structure, HDP protein and its two truncated variants. c,Recombinantly expressed and purified HDP proteins on a 12% Coomassiestained gel under reducing conditions. d, Immunoblot of purifiedproteins with anti-HDP antibodies.

FIG. 20A-D. Circuitous transport and delivery of HDP to the foodvacuole. a, HDP is secreted into the cytosol of infected erythrocytes(arrowhead) in early ring stages before any Hz could be detected insidethe parasite. b, After secreting it into the host cell cytosol, parasiteintakes HDP through the cytostome c, HDP could be found in transportvesicles destined to the food vacuole. d, Transport vesicles deliver HDPto the food vacuole where it was present in close proximity of Hzcrystals. cyt, cytostome; fvm, food vacuole membrane; fv, food vacuole;hz, hemozoin; hdp, heme detoxification protein; hb, hemoglobin; nu,nucleus; par, parasite; ppm, parasite plasma membrane; pvm,parasitophorous vacuole membrane; irbc, infected red blood cell; rbcm,RBC membrane; tv, transport vesicle. Bar, 0.5 μm.

FIG. 21A-C. HDP is transported to food vacuole along with hemoglobin. a,HDP(18 nm particles) was found in the cytosol of infected cells. Insetb, HDP is being internalized along with hemoglobin (12 nm particles).Inset c, Transport vesicle ready to deliver both, HDP and hemoglobin tothe food vacuole. Bar, 0.5 μm.

FIG. 22. Comparison of Hemozoin production by HDP protein from P. vivaxand P. falciparum.

FIG. 23. Results of immunization of mice with either DNA encoding HDPfrom P. yoelii or with P. yoelii HDP protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention is based on the discovery of several surprisingproperties of a previously uncharacterized family of parasite proteins.The protein family has been designated “FRAP” for “Fasciclin RelatedAdhesive Protein”. Alternatively, the protein is denominated “HDP” for“Heme Detoxification Protein”. Herein “FRAP” and “HDP” designate thesame entity. This protein is expressed by Plasmodium and Theileriaparasites and is intimately involved in the onset of parasiticinfections. Hence, the FRAP (HDP) family of proteins, and the nucleicacids that encode them, are ideal targets for the treatment and/orprevention of certain parasitic diseases.

The initial FRAP protein was selected for study based on a systematicanalysis of the genome of Plasmodium falciparum using a combination ofin-silico algorithms, microarray and proteomic techniques. This processis described in detail in Example 1 of the Examples section. The studypredicted that FRAP should be expressed on the surface of the P.falciparum sporozoite, and thus would be involved in early interactionsbetween the sporozoite and host cells, making it an attractive targetfor therapeutic intervention. These predictions have been confirmed.FRAP protein is present in micronemes, a specialized secretory organellethat transports proteins to the surface of the Plasmodium sporozoite.FRAP and an 87 amino acid polypeptide derivative, FRAP2 (amino acidsequence, SEQ ID NO: 25; nucleic acid sequence, SEQ ID NO: 26; FIG. 15)bind to liver cells, thereby preventing sporozoite invasion. Further,antibodies specific for FRAP2 also prevent sporozoite invasion of livercells. A thirty-two amino acid sequence that is recognized by theseantibodies, encodes the inhibitory epitope and is common to the FRAPfamily of proteins (TRSGGLRKPQKVTNDPESINRKVYWCFEHKPV, SEQ ID NO: 24),has also been discovered. This sequence shows 100% sequence homology and87.5% sequence identity within the Plasmodium genus. In addition, theenzymatic activity of FRAP has been elucidated. FRAP catalyzes theneutralization of toxic heme into non-toxic hemozoin, making thisprotein a highly significant target for inhibitory drug therapy.

Herein we describe the application of these discoveries to theprevention and treatment of parasitic diseases. For example, FRAPproteins and various derivatives of FRAP proteins, including theantigenic epitope, and the nucleic acids that encode them, are useful asvaccine components. In addition, the inhibition of FRAP proteins ornucleic acids that encode them (e.g. by compounds that bind to theactive site of the protein, or by RNA silencing) also provides astrategy for therapeutic intervention in parasitic disease. Further, theinvention provides diagnostic tools related to the detection ofparasites harboring either the FRAP protein or nucleic acids encodingFRAP. Further, the invention provides methods and compositions forinhibiting the ability of HDP to detoxify heme, i.e. to convert heme tohemozoin. Thus, the methods and compositions are useful for thetreatment or prevention of diseases caused by Plasmodium and Theileriaparasites. These and other aspects of the invention are discussed indetail below.

The FRAP protein that was first identified in (originated from) P.falciparum and is represented by SEQ ID NO: 1 (see FIG. 1). The proteinis encoded by the nucleic acid sequence represented by SEQ ID NO: 2(FIG. 2). However, the FRAP family of proteins is not limited to thoseoriginating from P. falciparum. FRAP orthologs from Plasmodium speciesother than P. falciparum have been identified, for example, FRAPorthologs from human (P. vivax) simian (P. knowlesi, P. reichenowi),avian (P. gallinaceum) and rodent (P. berghei, P. yoelii and P.chaubaudi) malaria parasites. Overall, FRAP has extremely high sequencehomology across the Plasmodium genus and the region encoding theinhibitory epitope identified in P. falciparum protein is very highlyconserved in all known FRAP orthologs. Furthermore, polymerization ofhuman heme into hemozoin by FRAP from rodent malaria parasite P. yoeliihas been demonstrated. Therefore, FRAP sequences between differentspecies of the parasites are functionally interchangeable and transgenicmalaria parasites expressing the FRAP sequence from any member of thePlasmodium genus can be utilized for human malaria drug and for vaccinedevelopment. In addition, FRAP orthologs present in many related speciessuch as Theileria may also be utilized for use in drug and vaccinedevelopment for the diseases they cause, e.g. bovine tropicaltheileriosis (Preston et al., Innate and adaptive immune responsesco-operate to protect cattle against Theileria annulata. ParasitolToday. 1999 July; 15(7):268-74). All such orthologs, examples of whichare given in FIG. 1, are encompassed by the present invention. Thenucleic acids that encode some exemplary FRAP proteins are presented inFIG. 2.

Those of skill in the art will recognize that a FRAP protein need nothave an exact sequence as depicted in FIG. 1 in order to be suitable foruse in the practice of the present invention. Rather, the invention alsoencompasses variants (derivatives) of such proteins. The term “protein”as used herein refers to sequences of about 100 or more amino acids; and

the term “polypeptide” refers to sequences of about 100 amino acids orless, although these terms may be used interchangeably. (Shortersequences, e.g. about 35 or fewer amino acids, will generally bereferred to as peptides.) Variants or derivatives of FRAP proteins maybe isolated from nature or be purposefully constructed. The primarysequence of such a variant or derivative may differ from the originalsequence (e.g. as represented in FIG. 1) in any of several ways,including the following: conservative amino acid substitutions;non-conservative amino acid substitutions; truncation by, for example,deletion of amino acids at the amino or carboxy terminus, or internallywithin the molecule; or by addition of amino acids at the amino orcarboxy terminus, or internally within the molecule (e.g. the additionof a histidine tag for purposes of facilitating protein isolation, thesubstitution of residues to alter solubility properties, the replacementof residues which comprise protease cleavage sites to eliminate cleavageand increase stability, the replacement of residues to form a convenientprotease cleavage site, the addition or elimination of glycosylationsites, and the like, for any reason). Such variants may be naturallyoccurring (e.g. as the result of natural variations between species orbetween individuals, or as a result of different expression systems usedto produce the amino acid sequence, etc.); or they may be purposefullyintroduced (e.g. in a laboratory setting using genetic engineeringtechniques). The amino acid sequences may be in a variety of forms,including a neutral (uncharged) forms, or forms which are salts, and maycontain modifications such as glycosylation, side chain oxidation ordeamidation, phosphorylation and the like. Also included are amino acidsequences modified by additional substituents such as glycosyl units,lipids, or inorganic ions such as phosphates, as well as modificationsrelating to chemical conversions or the chains, such as oxidation ofsulfhydryl groups.

All such variants of the amino acid sequences disclosed herein areintended to be encompassed by the teachings of the present invention,provided the variant protein/polypeptide displays sufficient identity tothe original sequence as disclosed herein, or an amino acid sequencethat can be translated from a nucleic acid sequence disclosed herein.Preferably, amino acid identity will be in the range of about 50 to100%, and preferably about 60 to 100%, or more preferably about 70 to100%, or even more preferably about 80 to 100%, or most preferably about90 to 100%, or even about 95 to 100%, of the disclosed sequences. Theidentity is with reference to the portion of the amino acid sequencethat corresponds to the original amino acid sequence as translateddirectly from the nucleic acid sequences disclosed herein, i.e. notincluding additional elements that might be added, such as sequencesadded to form chimeric proteins, histidine tags, etc. Those of skill inthe art are well acquainted with the methods available for determiningthe identity between amino acid sequences, for example, FASTA, FASTP,the BLAST suite of comparison software, ClustalW, Lineup, Pileup, ormany other alignment software packages.

In addition, such protein/polypeptide variants retain at least about 50to 100% or more of the activity of the original polypeptide, andpreferably about 60 to 100% or more, or more preferably about 70 to 100%or more, or even more preferably about 80 to 100% or more, and mostpreferably about 90 to 100% or more of the activity of the originalsequence. By “activity” we mean the activity or role of the amino acidsequence in the parasite from which is was isolated, which may includebut is not limited to: characteristic enzyme activity, activity as astructural component, role as a membrane component, binding activity,etc.

The peptides, polypeptides and proteins of the present invention aregenerally provided as recombinant molecules, although the amino acidsequences may also be produced synthetically via known peptide synthesistechniques. The peptides, polypeptides and proteins of the presentinvention are provided in a substantially purified form, i.e. they aregenerally free of extraneous materials (such as other proteins, nucleicacids, lipids, cellular debris, etc.) and will generally be at leastabout 75% pure, preferably about 85% pure, and most preferably at leastabout 90-95% or more pure, as would be understood by one of ordinaryskill in the art.

In general, the proteins and polypeptides of the invention are producedin recombinant expression systems. In a preferred embodiment of thepresent invention, the recombinant system is an E. coli recombinantsystem. However, they may also be produced in a variety of otherrecombinant expression systems. For example, yeast, insect cells (usingfor example, a baculovirus expression vector), plant cells (e.g.tobacco, potato, corn, etc.), transgenic animals, or mammalian cellculture systems can be used for expression of recombinant proteins. Anyappropriate expression system that suitably produces the proteins andpolypeptides of the invention may be used in the practice of theinvention. Such systems and their use for the production of recombinantproteins are well known to those of ordinary skill in the art.

The invention also provides antigenic peptides, in particular anantigenic epitope common to the FRAP family of proteins. The epitope hasthe amino acid sequence TRSGGLRKPQKVTNDPESINRKVYWCFEHKPV (SEQ ID NO:24). Some modification of this sequence may be tolerated withoutcompromising the antigenicity of the sequence. Those of skill in the artwill recognize that peptides may be obtained by several means, includingbut not limited to chemical synthesis methods, production using geneticengineering techniques, enzymatic digestion of larger polypeptides, etc.The particular source of a peptide is not a crucial feature of theinvention. In a preferred embodiment, the peptide will be chemicallysynthesized. In some embodiments of the invention, the FRAP epitope willbe used as an antigen in combination with at least one other knownparasite antigenic epitope. For example, genetic engineering techniquesmay be employed to construct chimeric polypeptides or proteinscontaining two or more of such epitopes on the same molecule.Alternatively, separate preparations of the peptidic epitopes may beprepared and mixed into a single solution, for example, to beadministered as a vaccine.

In addition to utilizing FRAP proteins, polypeptides and peptides, thepresent invention also encompasses use of the nucleic acids that encodesuch amino acid sequences. Exemplary DNA sequences that encode FRAPproteins are given in FIG. 2A-J. The nucleic acids may be used as atool, e.g. to produce a protein. Alternatively, the nucleic acidsequences themselves may be used in certain aspects of the invention,e.g. as components of DNA vaccines, or for gene silencing applications(see below). Those of skill in the art will recognize that many variants(derivatives) of such sequences may exist in nature or be constructedwhich would still be suitable for use in the practice of the presentinvention. For example, with respect to the translation of amino acidsequences from the nucleic acid sequences, due to the redundancy of thegenetic code, more than one codon may be used to code for an amino acid.Further, as described above, changes in the amino acid primary sequencemay be desired, and this would necessitate changes in the encodingnucleic acid sequences. In addition, those of skill in the art willrecognize that many variations of the nucleic acid sequences may beconstructed for purposes related to other aspects of the invention, forexample: for cloning strategies (e.g. the introduction of restrictionenzyme cleavage sites for ease of manipulation of a sequence forinsertion into a vector, for rendering the sequence compatible with thecloning system vector or host, for enabling fluorescent or affinitylabeling technologies, etc.), for purposes of modifying transcription(e.g. the introduction of specific promoter or enhancer sequences,insertion or deletion of splice signals, for enhancing or negativelyregulating transcription levels, for regulating polyadenylation, forcontrolling termination, and the like), or for modification of active orinactive domains, for elimination or modification of certain activitiesor domains, for optimizing expression due to codon usage or othercompositional biases, for addition of immunologically relevant(enhancing or inhibiting) sequences or for any other suitable purpose.All such variants of the nucleic acid sequences encoding the proteins,polypeptides and peptides disclosed herein are intended to beencompassed by the present invention, provided the sequences displayhomology in the range of about 50 to 100%, and preferably about 60 to100%, or more preferably about 70 to 100%, or even more preferably about80 to 100%, or most preferably about 90 to 100% or about 95 to 100% tothe disclosed sequences. The homology is with reference to the portionof the nucleic acid sequence that corresponds to the original sequence,and is not intended to apply to additional elements such as promoters,vector-derived sequences, restriction enzyme cleavage sites, etc.derived from other sources. Those of skill in the art arewell-acquainted with methods to determine nucleic acid similarity oridentity using simple software alignment tools such as FASTA, the BLASTsuite of programs, CLUSTAL W, Lineup, Pileup (GCG), or many others.

In addition, the nucleic acids are not limited to DNA, but are intendedto encompass other nucleic acids as well, such as mRNA, RNA-DNA hybrids,and various modified forms of DNA and RNA known to those of skill in theart. For example, for use in vivo, nucleic acids may be modified toresist degradation via structural modification (e.g. by the introductionof secondary structures, such as stem loops, or via phosphate backbonemodifications, etc.). Alternatively, the nucleic acids may includephosphothioate or phosphodithioate rather than phosphodiesteraselinkages within the backbone of the molecule, or methylphosphorothiateterminal linkages. Other variations include but are not limited to:nontraditional bases such as inosine and queosine; acetyl-, thio- andsimilarly modified forms of adenine, cytidine, guanine, thymine anduridine; stabilized nucleic acid molecules such as nonionic DNA analogs,alkyl- and aryl phosphonates; nucleic acid molecules which contain adiol, such as tetrahyleneglycol or hexaethyleneglycol, at either or bothtermini; etc. Further, the nucleic acid molecules may be either singleor double stranded, or may comprise segments of both single and doublestrand nucleic acid.

In the course of practicing the invention, FRAP-related nucleic acidmolecules may be cloned into one of many suitable vectors. In someembodiments, vectors containing nucleic acid sequences (e.g. DNA) thatencode the amino acid sequences of the invention will encode a singleprotein, polypeptide, or peptide. However, this need not always be thecase. Such vectors may contain DNA encoding more than one amino acidsequence, either as separate, discrete sequences, or combined into asingle chimeric sequence. For example, in the case of an expressionvector, two or more nucleic acids according to the invention may bepresent in the vector, and the nucleic acids may be expressedseparately, resulting in the translation of one amino acid sequence foreach nucleic acid. Alternatively, a single polypeptide chain containingmore than one amino acid sequence of the invention, or portions of morethan one amino acid sequence of the invention, may be combined intandem. For example, one or more highly antigenic proteins or regions ofproteins of the invention may be expressed as a chimera from a singleDNA sequence. Alternatively, the amino acid sequences of the inventionmay be expressed as part of a chimeric protein comprising amino acidsequences from another source, e.g. antigenic sequences known to beuseful as adjuvants (e.g. PADRE [and other Pan-DR T helper cellepitope], hepatitis B core antigen, DNA sequences CPG, other chemokines,CTB or cholera toxin B subunit, Ricin B and other plant toxin subunits,LPS or lipopolysaccharide, KLH [key hole limpet hemocyanin], Freund'scomplete and Freund's incomplete adjuvant, and many other reagents,etc.), sequences that permit targeting of the protein to a specificlocation within the cell (e.g. nucleus, nucleolus or nuclear membrane,mitochondrion/mitosome/mitochondria-like organelle, membrane,endoplasmic reticulum, golgi, rhoptry, dense granules, calcisomes oracidocalcisomes, and other subcellular organelles compartments, etc.).

One application of the present invention is the provision of vaccinesthat provide immunity to disease caused by parasites such as Plasmodium.By “immunity” we mean that administration to an individual of one ormore proteins, polypeptides or peptides of the invention, or nucleicacids encoding them, either alone or in combination with other antigenicentities prevents the development of disease symptoms in that individualafter exposure to or infection by a parasite. Alternatively, the diseasesymptoms that develop in the individual may be milder than those thatwould otherwise develop in, for example, a matched control individual.Those of skill in the art are well acquainted with the use and meaningof “controls” when comparing results of individuals or populations thathave been exposed to different variables (e.g. vaccinated or not). Inparticular, the inhibitory epitope peptide of the invention may be usedin combination with one or more other antigenic epitopes for theproduction of a multicomponent vaccine. Such a vaccine addressesprevious lackluster vaccine performance by presenting several highlyimmunogenic epitopes to the immune system of a vaccinated individual ina single preparation. This type of vaccine closely mimics the natural invivo presentation of antigens on the surface of a parasite, and thuselicits a robust immune response.

According to an embodiment of the invention, the vaccine may either beprophylactic (i.e. to prevent or attenuate symptoms of infection) ortherapeutic (i.e. to treat disease after infection). Such vaccinescomprise one or more of: immunizing antigen(s), immunogen(s),polypeptide(s), protein(s) and nucleic acid(s) from the FRAP family (asdescribed herein), usually in combination with “pharmaceuticallyacceptable carriers,” which include any carrier that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,lipid aggregates (such as oil droplets or liposomes), and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Additionally, these carriers may function as immunostimulatingagents (“adjuvants”). Furthermore, the antigen or immunogen may beconjugated to a bacterial toxoid, such as a toxoid from diphtheria,tetanus, cholera, H. pylori, etc. pathogens. Preferred adjuvants toenhance effectiveness of the composition include, but are not limitedto: (I) aluminum salts (alum), such as aluminum hydroxide, aluminumphosphate, aluminum sulfate, etc; (2) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: thesubunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE (see below), although not required)formulated into submicron particles using a microfluidizer such as Model100Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP (see below) either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) Ribi™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS(Detox™); (3) saponin adjuvants, such as Stimulon™ (CambridgeBioscience, Worcester, Mass.) may be used or particles generatedtherefrom such as ISCOMs (immunostimulating complexes); (4) CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA); (5)cytokines, such as interleukins (eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7,IL-12, etc.), interferons (eg. gamma interferon), macrophage colonystimulating factor (M-CSF), tumor necrosis factor, etc; and (6) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the composition. Alum and MF59™ are preferred.

The immunogenic compositions (eg. the immunizingantigen/immunogen/polypeptide/protein/nucleic acid, pharmaceuticallyacceptable carrier, and adjuvant) typically will contain diluents, suchas water, saline, glycerol, ethanol, etc. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles. Typically,the immunogenic compositions are prepared as injectables, either asliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection may also beprepared. The preparation also may be emulsified or encapsulated inliposomes for enhanced adjuvant effect, as discussed above underpharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the antigenic or immunogenic polypeptides, as wellas any other of the above-mentioned components, as needed. By“immunologically effective amount”, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for eliciting the production of antibodies, foreliciting a cellular immune response, (or both), and/or for treatment orprevention of disease. This amount varies depending upon the health andphysical condition of the individual to be treated, the taxonomic groupof individual to be treated (e.g. nonhuman primate, primate, etc.), thecapacity of the individual's immune system to synthesize antibodies, thedegree of protection desired, the formulation of the vaccine, thetreating doctor's assessment of the medical situation, and otherrelevant factors. It is expected that the amount will fall in arelatively broad range that can be determined through routine trials.The immunogenic compositions are conventionally administeredparenterally, eg. by injection, either subcutaneously, intramuscularly,intranasally, or transdermally/transcutaneously. Additional formulationssuitable for other modes of administration include oral and pulmonaryformulations, suppositories, and transdermal applications. Dosagetreatment may be a single dose schedule or a multiple dose schedule. Thevaccine may be administered in conjunction with other immunoregulatoryagents. As an alternative to protein-based vaccines, DNA vaccination maybe employed [eg. Robinson & Torres (1997) Seminars in Immunology9:271-283; Donnelly et al. (1997) Annu Rev Immunol 15:617-648].

Vaccines can be composed of live, attenuated or killed organisms, orchemically inactivated toxins (toxoids), against which the body canraise an effective immune response, leading to effective protectionagainst the live agent or active toxins produced during the infection.Combination vaccines make it possible to immunize individuals againstmultiple pathogens at a time. Examples of combination vaccines are DTaP(Diphtheria, Tetanus, combined with acellular Pertussis) or MMR(Measles, Mumps, and Rubella). Conjugated vaccines, such as PCV(Pneumococcal Conjugated Vaccine) provide better immunization ofinfants. In conjugated vaccines polysaccharide antigens are chemicallylinked to protein antigens which provide a better stimulus for theimmature immune system. Through the use of recombinant DNA technology itis possible to isolate and express individual genes or combinations ofgenes, encoding antigens from pathogens and produce vaccines byfermentation. Recent advances in genomics and proteomics of(re-)emerging pathogens will enable entirely new generations of vaccinebased on identification of surface proteins. Table 1 lists common typesof vaccines in current use or in development, and some importantattributes. TABLE 1 Vaccine types in current use and development TypeVaccine Advantages Disadvantages Live, attenuated Measles, mumps,Produce a strong Remote possibility vaccines rubella, polio (Sabinimmune response; that the live microbe vaccine), yellow often givelifelong could mutate back to fever immunity with one a virulent form;or two doses must be refrigerated to stay potent Inactivated or Cholera,flu, Safer and more Produce a weaker “killed” vaccines hepatitis A,Japanese stable than live immune response encephalitis, plague,vaccines; don't than live vaccines; polio (Salk vaccine), requirerefrigeration; usually require rabies more easily stored, additionaldoses transported Toxoid vaccine Diphtheria, tetanus Teaches immuneProtect only against system to fight off deleterious effect of bacterialtoxins; toxin, but do not often easy to provide protection produce frompathogen Subunit vaccines Hepatitis B, Targeted to very When developinga pertussis, specific parts of the new vaccine, pneumonia causedmicrobe; fewer identifying the best by Streptococcus antigens, so lowerantigens can be pneumoniae chance of adverse difficult and timereactions consuming Conjugate vaccines Haemophilus Allow infantinfluenzae type B, immune systems to pneumonia caused recognize certainby Streptococcus antigens pneumoniae DNA vaccines In development Producea strong Still in experimental antibody and stages cellular immuneresponse; relatively easy and inexpensive to produce Recombinant vectorIn development Closely mimic a Still in experimental vaccines naturalinfection, stages stimulating a strong immune responseSource: Understanding Vaccines: What they are, how they work. U.S.DHHS/NIH/NIAID, NIH Publication No. 03-4219, 2003.

Most vaccines in Table 1 are administered by subcutaneous orintramuscular injection. The oral route of administration isoccasionally used in case of Oral Polio Vaccine. New vaccine technologyis being developed to produce vaccines that (i) generate stronger andbroader immunity, (ii) meet more stringent safety and qualityrequirements, and (iii) that have greater ease of delivery at lowercost. Therefore, a significant amount of research is ongoing to developnew delivery methods and adjuvants. For effective immunization mostvaccines are delivered using adjuvants. Adjuvants are emulsions orformulations, often containing lipids or aluminum salts, which providefor slow release of the antigen into the plasma, and also stimulate theimmune response in ways that are not fully understood. Slow release ofthe antigen is also important to prevent metabolism and removal from theplasma prior to the initiation of the immune response. Delivery ofantigen to the cells that participate in antigen presentation,macrophages and dendritic cells, is also improved by the use ofadjuvants. Table 2 lists a number of commonly used adjuvants and newadjuvant delivery methods in development. TABLE 2 Commonly usedadjuvants and new products in development. Adjuvant Category New productor method Comments/Examples Gel type Aluminium hydroxide/phosphateImprove delivery to APCs and Calcium phosphate secondary lymphoid organsMicrobial Muramyl dipeptide (MDP) Bacterial exotoxins Cholera toxin (CT)Endotoxin based adjuvants Escherichia coli heat labile toxin (LT)Monophosphoryl lipid A (MLA) Particulate Biodegradable polymermicrospheres Immuno-stimulatory complexes (ISCOMs) Liposomes Oilemulsion/ Freunds incomplete adjuvant Animal experimental uses onlysurfactant Microfluidized emulsions MF59 (Squalene), SAF Saponins Qs-21Synthetic Muramyl peptide derivatives Murabutide, Threonyl-MDP Non-ionicblock co-polymers L121 Polyphosphazene (PCPP) Cytokines Interleukin-2,-12 Molecules secreted by GM-CSF macrophages or dendritic cellsInterferon gamma that stimulate the inflammatory and immune responseGenetic Genes encoding cytokines or co- IL-12, IL-2, IFNg, CD40Lstimulatory molecules delivered by plasmidsSources: Progress in Immunologic Adjuvant Development 1982-2002, TheJordan Report 2002, US DHHS/NIH/NIAID, and the website located atwww.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf.

New physical administration methods being developed include delivery byinhalation, oral delivery, or transdermal delivery. Inhalation deliveryincludes intranasal delivery for delivery to the upper respiratorytract, which is being used in FluMist (influenza vaccine) or otherpowder or particle based methods to deliver immunization to the lowerrespiratory tract. Oral delivery includes new formulations to allowantigens to pass through the stomach and intestinal tract without acidor protease inactivation. New methods of oral delivery include ediblevaccines, where plants such as potatoes, tomatoes, or bananas aregenetically engineered to express the antigen in parts of the plant thatare consumed by humans. New transdermal delivery methods that avoidinjection are being explored as well. However the large size (highmolecular weight) of the antigen(s) usually is a limitation for thisdelivery method. A relatively new delivery method is expression ofantigens in a strain of virus or a bacterium that is not naturallypathogenic, or is made avirulent either through mutation or geneticengineering. Attenuated viruses such as polio, or bacteria such asVibrio cholerae and Salmonella typhi, are being explored as deliveryvehicles.

Production methods for vaccines vary with the type of vaccine. Live,attenuated or killed virus vaccines are produced in mammalian cellculture. In the latter case virus particles are killed by chemicalinactivation, heat or radiation. A major concern of mammalian cellculture based production methods is contamination with other pathogens,specifically retroviruses such as HIV, or other as of yetuncharacterized mammalian viruses. Influenza vaccine is produced eitherthrough cell culture or growth of virus in fertilized chicken eggs,followed by purification from the yolk. Live, attenuated or killedbacterial vaccines are produced by microbial fermentation. Concerns withthis method are contamination with other micro-organisms (bio-burden),or presence of bacterial endo- or exo-toxins that can cause anaphylacticshock. Toxoid vaccines, such as diphtheria or tetanus vaccines, areproduced by microbial fermentation and harvesting of the exo-toxins fromthe culture medium. Toxoid vaccines can also be produced withrecombinant DNA technology, followed by purification of the recombinantprotein. Conjugated vaccine components are produced through multiplemethods. The polysaccharide component is harvested from bacteria grownin culture, and the protein component of the antigen can be producedthrough fermentation or recombinant DNA technology. The conjugation stepis done through a chemical reaction. Subunit vaccines, existing ofspecific protein antigens (or combinations) are made throughfermentation or recombinant DNA technology. Other transgenic productionmethods, such as expression in the milk of transgenic animals, orproduction in genetically engineered plants, are being explored forsubunit vaccines as well. DNA vaccines are produced using recombinantDNA technology. Vector vaccines are produced through genetic engineeringof the vector, i.e. to produce the antigens of interest, and eithermicrobial fermentation or mammalian cell culture.

In particular, with respect to DNA vaccines, U.S. Pat. No. 6,214,804(Felgner, et al., 2001, the complete contents of which is herebyincorporated by reference) describes the induction of a protectiveimmune response in a mammal by injecting a DNA sequence. Methods fordelivering an isolated polynucleotide to the interior of a cell in avertebrate are provided. The methods can be used to deliver atherapeutic polypeptide to the cells of the vertebrate, to provide animmune response upon in vivo translation of the polynucleotide, todeliver antisense polynucleotides, to deliver receptors to the cells ofthe vertebrate, or to provide transitory gene therapy.

In addition, U.S. Pat. No. 6,923,958 (Xiang et al., 2005, the completecontents of which is hereby incorporated by reference) describes DNAvaccines encoding carcinoembryonic antigen (CEA) and a CD40 ligand andmethods of their use. The DNA vaccine is effective for eliciting animmune response against cells that present a carcinoembryonic antigen,and could be incorporated in a delivery vector such as an attenuatedlive bacterium or virus, or a liposome carrier. Alternatively, the DNAvaccine is administered orally to a mammal, such as a human, to elicitan immune response against CEA presenting cells such as colon cancercells. The mammal may be further treated with recombinant antibodyfusion proteins to enhance the immune response effectiveness of thevaccine.

Another embodiment of the invention provides antibodies specific forFRAP proteins, polypeptides and peptides. As used herein, the term“antibody” refers to a polypeptide or group of polypeptides composed ofat least one antibody combining site. An “antibody combining site” isthe three-dimensional binding space with an internal surface shape andcharge distribution complementary to the features of an epitope of anantigen, which allows binding of the antibody with the antigen.“Antibody” includes, for example, vertebrate antibodies, hybridantibodies, chimeric antibodies, humanised antibodies, fully humanantibodies, altered antibodies, univalent antibodies, Fab proteins andfragments, and single domain antibodies. Antibodies to the polypeptidesand peptides of the invention, both polyclonal and monoclonal, may beprepared by conventional methods that are well-known to those of skillin the art. If desired, the antibodies (whether polyclonal ormonoclonal) may also be labeled using conventional techniques.

Antibodies for therapeutic applications for the prevention or treatmentof malarial disease, or diagnostic applications in the detection ofparasite infection, can be made by standard methods. In most cases theantibodies will be of monoclonal origin, and either produced in rats ormice.

Protein for immunization is made by recombinant methods. Any of theproteins from the group of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17,and 19, or portions thereof, can be produced by cloning thecorresponding DNA sequences of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16,18, and 20, or portions thereof, in recombinant protein expressionvectors. Protein can be produced in this manner in E. coli, yeast,fungi, plants, mammalian, or insect cells. It is obvious that thepreferred protein used for immunization is from the Plasmodium speciesthat infect humans, i.e. SEQ ID NOS: 1 and 7. However, in principle SEQID NOS: 3, 5, 9, 11, 13 and 15, could also be used to generateantibodies that are effective as therapeutics or diagnostic tools.Immunization material for short peptides and small proteins can also bemade through chemical synthesis.

For example the 8-mer peptide represented by SEQ ID NO: 37 may beencoded by: ACCAACGACCC AGAAAGTATAAAT (SEQ ID 38), or other sequences;and the 32-mer peptide represented by SEQ ID NO: 24 may be encoded by:ACACGAAGTGGCGGTTTAAGAAAACCTCAAAAGG TAACCAACGACCCAGAAA GTATAAATAGAAAAGTATATTGGTGTTTTGAACATAA GCCTGTA (SEQ ID 39), or othersequences. Alternatively, the these peptides may be chemicallysynthesized.

Expressed protein can be purified with standard HPLC and otherchromatographic methods, in quantities and sufficient purity to beinjected in the mice or standard rats. Rats or mice are injected in thepresence of adjuvants, and in a standard schedule of injections andboosters, in order to generate a vigorous immune response. In order tomake monoclonal antibodies, spleen cells are harvested from the animalsand fused with immortalized cell lines. Numerous immortalized cell linesare screened for their ability to secrete antibodies that bind theoriginal antigen used in immunizations. Positive cell lines are purifiedand cloned, and their antibodies are characterized and screened toidentify antibodies that have strong binding characteristics. Uponidentification of such cell lines, the antibody genes are cloned,sequenced and can be used to engineer mammalian cell culture strains forhigh level production.

In order to avoid a human immune response against the therapeuticantibody, the sequence of the monoclonal antibody is modified to mostclosely resemble the sequence of native human antibodies. This is doneby recombinant DNA methods, through selective replacement of thesignificant portions of the munine antibody light and heavy chainsequences with human sequences (chimeras), or through replacement ofalmost all of the non-variable sections of the murine antibody light andheavy chains, with those from human antibody chain conserved sequences,while maintaining the original rat or mouse sequence of thehyper-variable domain which is responsible for antigen recognition andbinding (‘CDR grafting’ or ‘humanization’). For example U.S. Pat. No.6,500,931 describes the method of humanizing antibodies.

Alternatively, fully human monoclonal antibodies can be made in micedirectly, when these mice are engineered to produce only human antibodychains. For example the technology practiced by companies such asAbgenix Inc. [XenoMouse technology, U.S. Pat. No. 6,657,103], MedarexInc. and GenMab A/S [HuMab Mouse or UltiMAB technology; WO2005023177]can be used. Purified proteins as described above are used to immunizesuch engineered mice. Monoclonals produced in this manner are produced,screened and characterized in the standard manner. Fully humanantibodies can also be produced using phage display methods by screeningagainst human antibody phage display libraries. For example technologiespracticed by companies such as Cambridge Antibody Technology [U.S. Pat.No. 5,969,108 and U.S. Pat. No. 6,172,197] and others, can be used toidentify fully human antibodies in this manner. Phage display screeninghas an added advantage in that the process does not rely on animalimmunization. The genes for fully human antibodies produced usingengineered mice, or identified through phage display, can be isolated,sequenced and cloned for expression in mammalian cell lines for highlevel expression using standard methods.

Patents describing this technology in detail are incorporated herein byreference.

Such antibodies may be used, for example, for affinity chromatography,immunoassays, and for distinguishing or identifying parasite proteins orportions thereof. In a preferred embodiment of the invention, suchantibodies may be used therapeutically, e.g. for administration topatients suffering from a parasitic disease such as malaria, orprophylactically in order to prevent a parasitic disease in patients atrisk for developing the disease.

In yet another embodiments of the invention cells or cell linescontaining the nucleic acids and/or the amino acid sequences of theinvention as described herein. For example, the cell may be a host cellthat harbors one or more vectors containing nucleic acid sequences usedin the invention (e.g. DNA or RNA) and/or amino acid sequences of theinvention translated from such vectors. Such cells may contain multiplevectors, and the vectors may be the same or different. Further, thecells may be either in vitro or in vivo. The invention also comprehendspharmaceutical compositions and their use. The pharmaceuticalcompositions can comprise one or more proteins, polypeptides, peptides,antibodies, or nucleic acids according to the invention, or combinationsof these. In addition, the compositions may include compounds thatinhibit the interaction of HDP and heme, thereby preventing or vitiatingthe ability of HDP to detoxify heme, e.g. to form hemozoin from heme.The pharmaceutical compositions comprise a therapeutically effectiveamount of such molecules. The term “therapeutically effective amount” asused herein refers to an amount of a therapeutic agent that issufficient to treat, ameliorate, or prevent a disease or condition, orto exhibit a detectable therapeutic or preventative effect. The effectcan be detected by, for example, chemical markers or antigen levels.Therapeutic effects also include reduction of physical symptoms of theparasitic disease. The precise effective amount for a subject willdepend upon several parameters, including the subject's size, generalhealth, gender, age, etc., and the therapeutics or combination oftherapeutics selected for administration. Thus, it is not useful tospecify an exact effective amount in advance. However, the effectiveamount for a given situation can be determined by routineexperimentation and is within the judgement of those of skill in theart, e.g. a physician. For purposes of the present invention, aneffective dose will be from about 0.01 mg/kg to 50 mg/kg or about 0.05mg/kg to about 10 mg/kg of active, therapeutic agent.

A pharmaceutical composition may also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, inhibitory compounds, and othertherapeutic agents. Suitable carriers may be large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andinactive virus particles. Such carriers are well known to those ofordinary skill in the art. Pharmaceutically acceptable salts can be usedtherein, for example, mineral acid salts such as hydrochlorides,hydrobromides, phosphates, sulfates, and the like; and the salts oforganic acids such as acetates, propionates, malonates, benzoates, andthe like. A thorough discussion of pharmaceutically acceptableexcipients is available in Remington's Pharmaceutical Sciences (MackPub. Co., N.J. 1991).

In addition, pharmaceutically acceptable carriers in therapeuticcompositions may contain liquids such as water, saline, glycerol andethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, adjuvants, and the like,may be present in such vehicles. Typically, the therapeutic compositionsare prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection may also be prepared. Liposomes are included withinthe definition of a pharmaceutically acceptable carrier.

Once formulated, the compositions of the invention are administered tothe subject. The subjects to be treated may be animals; in particular,human subjects can be treated. Direct delivery of the compositions willgenerally be accomplished by injection, either subcutaneously,intraperitoneally, intravenously or intramuscularly or delivered to theinterstitial space of a tissue. Other modes of administration includeoral and pulmonary administration, suppositories, and intranasal,transdermal or transcutaneous applications (eg. see WO98/20734),needles, and gene guns or hyposprays. Dosage treatment may be a singledose schedule or a multiple dose schedule.

Yet another embodiment of the invention provides tools and methods forthe diagnosis of parasitic infections. Such tools include primerscontaining nucleotide sequences that specifically hybridize to nucleicacid sequences that are unique to FRAP. Hybridization of the primers tosuch a unique sequence permits amplification of the unique sequence (forexample, by polymerase chain reaction (PCR)), thus providing a means tospecifically identify the presence of FRAP in biological samples (blood,feces, sputum, urine, bronchoaveloar lavage, etc.). Amplification may bedirectly from the genome of the organism located in the sample, or fromRNA, e.g. mRNA.

By “primer” we mean a nucleotide sequence that hybridizes to anothernucleotide sequence of interest, the primer typically being a relativelyshort nucleotide sequence (e.g. from about 10 to about 100 base pairs)and the nucleotide sequence of interest typically being transcribed fromthe genome of an organism. PCR amplification techniques are well-knownto those of skill in the art. In general, two primers are selected thattarget sites that flank the sequence of interest (e.g. a gene encodingFRAP) for diagnostics or identification. These primers are designed torecognize only the target sequence; i.e., they will hybridize only tothe target sequence and to no other sequences. The primers generallyrange from 18-nucleotides in length (but can be longer or shorter), haveTm's (melting temperatures) that are selected to be compatible with bothamplification conditions and with specificity, have little or nointernal structure (stem-loop structures caused by internalcomplementarity), little or no ability to dimerize with themselves,little or no ability to dimerize with the other primer, have fewhomopolymeric stretches, etc. Many computer programs (e.g., Primer3,Oligo, etc.) are available for primer design. At times, an internalfluorescent probe is also included for specific use in even moresensitive and automated tests. The internal probe is fluorescentlylabeled such that it is specifically degraded and therefore fluorescesonly if it specifically hybridizes to the target sequence. Alternately,other fluorescent probes can be designed that only fluoresce uponbinding specifically to an amplified specific sequence. Thus, severalalternative approaches are available for the generation and detection ofspecific sequences amplified by PCR, and any of these can be applied fordiagnostic or identification purposes. (See, for example: Mullis, K., F.Faloona, S. Scharf, R. Saki, G. Horn, and H. Erlich. (1986) Specificenzymatic amplication of DNA in vitro: The Polymerase Chain Reaction.Cold Spring Harbor Symposia on Quantitative Biology 51: 263; Saiki, R.K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K.B. Mullis, and H. A. Erlich. (1988) Primer-directed enzymaticamplification of DNA with a thernostable DNA polymerase. Science239:487; Schutzbank T E, Stern H J. (1993) Principles and applicationsof the polymerase chain reaction. J Int Fed Clin Chem. 1993July;5(3):96-105; Erlich H A. (1999) Principles and applications of thepolymerase chain reaction. Rev Immunogenet. 1(2):127-34; Wang, A. M.,Doyle, M. V., and D. F. Mark. (1989) Quantitation of mRNA by thepolymerase chain reaction. Proc Natl Acad Sci USA. 1989 December;86(24): 9717-9721; Kawasaki, E. S., and A. M. Wang. (1989) Detection ofgene expression. In: Erlich, H. A., ed., PCR Technology: Principles andApplications of DNA Amplification. Stockton Press, Inc., New York, N.Y.,pp. 89-97; Dieter Klein (2002) Quantification using real-time PCRtechnology: applications and limitations. Trends in Molecular Medicine,8(6):257-260; Buck GE. (1996) The polymerase chain reaction: arevolutionary new procedure for the laboratory diagnosis of infectiousdisease. J Ky Med Assoc. Apr; 94(4):148-52.)

Because the primers are unique to FRAP, a positive amplification resultis indicative of the presence of FRAP in the biological sample, and thusof infection by a parasite whose genome encodes FRAP. Similar tests canbe carried out with antibodies specific for FRAP. In this case, apositive result indicates that the biological sample being testedcontains FRAP, and thus, by inference, the individual from whom thesample was obtained is infected with a parasite that produced FRAP.

The invention further provides methods for treating or preventing adisease caused by a Plasmodium or Theileria parasite in an individual inneed thereof. In one embodiment, this is accomplished by inhibiting oneor more interactions of heme and Heme Detoxification Protein (HDP).Typically, inhibition is brought about by the administration of one ormore compounds that inhibit one or more interactions of heme and HDP. Inother words, the ability of HDP to produce hemozoin from heme (i.e. todetoxify heme) is eliminated or impaired by administration of thecompound. Examples of diseases that can be treated in this mannerinclude but are not limited to malaria, East Coast Fever caused byTheileria parasites, etc. Exemplary compounds that may be used in suchmethods are listed in Table 11 in the Examples section below. One ormore compounds from one or more of these classes may be administered, ina quantity sufficient to prevent or ameliorate disease symptoms.

Those of skill in the art will recognize that the mechanism of action ofthe compounds that are administered can be any of many known or not yetelucidated types, and that the precise mechanism(s) will depend on thecompound(s) administered. For example, the compound may bind to the HDPenzyme and prevent the enzyme from binding to heme. Alternatively, thecompound may bind to HDP and allow heme to also bind to HDP, but preventfurther catalysis and the production of hemozoin. For example, thecompound may bind at the active site or near the active site andsterically prevent the binding of heme; or the compound may bind at anallosteric site that influences (e.g. decreases) the activity of theenzyme; or the compound may cause heme to bind to HDP irreversibly orwith so great an affinity that the ability of HDP to detoxify heme iseliminated or attenuated. Alternatively, the compound may bind to heme.In this case, binding of the compound to heme may prevent the heme fromthen binding to HDP, or may allow the heme-compound complex to bind butnot be further processed to hemozoin. Those of skill in the art willrecognize that in all cases, the binding of compounds to HDP or to hememay be reversible or irreversible, realizing that all binding eventsinvolve an equilibrium distribution of bound and free agents. Thecriteria for the use of a compound in the present invention is that thecompound, regardless of its mechanism of action, decrease the productionof hemozoin from heme by at least about 10 to 25%, preferably from about25 to 50%, and more preferably from about 50 to 75%, or even from about75 to 10%.

Other possible mechanisms of action of the compounds that areadministered include but are not limited to: modification of a cellmembrane of the Plasmodium or Theileria parasite; inhibiting secretionof HDP from the Plasmodium or Theileria parasite, inhibiting transportof HDP to the food vacuole, the site of hemozoin formatin; by binding tofree heme (the substrate of HDP) and preventing its detoxification intoHemozoin; etc.

The administration of the compound(s) may be carried out by any suitablemeans, examples of which include but are not limited to orally,parenterally, sublingually, rectally, topically or with an inhalationspray.

In a preferred embodiment of the invention, the disease that isprevented or treated is malaria. In this case, the compound that isadministered may be administered in combination with one or moreadditional agents such as other antimalarial agents, agents forreversing antimalarial resistance, and various adjuvants. Administrationof one or more additional antimalarial agents or agents for reversingantimalarial resistance may occur prior to, concurrent with, orsubsequent to administration of the compound. Exemplary additionalantimalarial agents include but are not limited to quinolines, folicacid antagonists, sulfonamides, and antibiotics. An exemplary agent forreversing antimalarial resistance is an inhibitor of multidrugresistance. Exemplary adjuvants include but are not limited to thosewhich are suggested above for use in vaccine preparations, e.g. alum,etc.

The invention further comprehends pharmaceutical compositions comprisinga pharmaceutically acceptable carrier and an antimalarially effectiveamount of at least one compound. By “antimalarially effective amount” wemean that the compound is present in the composition in amount that,upon administration to an individual in need, prevents or lessens theoccurrence of symptoms associated with malaria in the recipient. Suchcompositions may include other active agents as well, e.g. adjuvants,other antimalarial agents (quinolines, etc.), agents that reverseresistance to malaria, etc.

The invention also provides method of inhibiting heme detoxification ina Plasmodium or Theileria parasite by preventing or attenuating theproduction of hemozoin by HDP in the parasite. Those of skill in the artwill recognize that various routes of inhibition may be effective. Forexample: inhibiting interaction of heme and HDP; preventing aninteraction of HDP or heme with cofactors; preventing dimerization ofHDP; preventing interaction of HDP or heme with lipids; and others.Exemplary cofactors, the interaction of which with HDP or heme may bedisrupted, include but are not limited to metal ions, natural ligandsand protein factors.

The invention also provides methods for identifying compounds thatinhibit HDP expression. The methods include the steps of a) contactingPlasmodium with a test compound and b) determining whether HDP isexpressed by the Plasmodium. Those of skill in the art will recognizethat there are several suitable methods to evaluate the outcome of suchtests, including but not limited to measuring mRNA that encodes HDP,measuring HDP protein production directly (i.e. detecting and measuringthe protein itself), etc.

The following Examples describe: the discovery and characterization ofthe novel FRAP protein family; the expression, localization andpurification of recombinantly expressed FRAP; the generation ofantibodies to FRAP2; experiments demonstrating the binding of FRAP toliver cells; prevention of sporozoite invasion of liver cells by FRAPand antibodies to FRAP2; discovery of the inhibitory epitope of FRAP;FRAP as a drug target; the use of FRAP in high throughput assays forhemozoin formation for screening novel antimalarials; siRNA mediatedinhibition of FRAP; the creation of FRAP variant attenuated parasitesfor use as whole organism vaccines; and the use of FRAP as a tool forhigh levels of expression and purification of recombinant proteins; thescreening of compounds that inhibit HDP; the results of in vivo testingof DNA that encodes HDP as a vaccine.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above and in the Examplessection below, but should further include all modifications andequivalents thereof within the spirit and scope of the descriptionprovided herein.

EXAMPLES Example 1 Discovery and Characterization of a Novel Plasmodiumfalciparum Protein Involved in Malaria Pathogenesis

Plasmodium sporozoites adhere to and invade host liver cells, leading tothe onset of malaria. Here we describe a novel, 205 amino acids long,Plasmodium falciparum protein involved in sporozoite-liver cellinteractions. Orthologs of this protein were identified in seven otherPlasmodium species, representing the four distinct phylogenetic clades,and the protein showed 60% sequence identity within the genus.Additionally, amino acids 88-205 have a 20% sequence identity tofasciclin 1, an ancient adhesive domain found in prokaryotes, plants andanimal proteins. The DNA encoding the protein was cloned, expressed inE. coli and the protein was purified to homogeneity. Immunoelectronmicroscopy showed that the protein was localized in the micronemes ofthe sporozoites. The protein contributes to sporozoite's adhesion andinvasion activities and antibodies raised against this protein canprevent >94% of P. falciparum sporozoites from invading liver cells,thus suggesting a role for this protein in malaria pathogenesis.Furthermore, we provide evidence that the protein exploits heparansulfate proteoglycans expressed on the liver cell surface as itsreceptor. Due to its role in host cell adhesion and the presence offasciclin 1 domain, we have named this protein as Fasciclin RelatedAdhesive Protein or FRAP. Our results show that FRAP is an excellenttarget for malaria vaccine development.

A bite by a parasite-infected mosquito delivers Plasmodium sporozoitesin the blood stream, which is followed by its entry into the livercells. A successful adhesion and invasion of liver cells by the parasitesets the stage for rapid multiplication, development and subsequentrelease of parasites in circulation, leading to the erythrocyticinfection and the clinical pathology associated with malaria. It iswidely believed that the host cell adhesion and invasion is a multistepprocess involving several parasitic proteins, many of which arecurrently not known. Of these, Circumsporozoite (CS) and SporozoiteSurface Protein-2/Thrombospondin-Related Anonymous Protein (SSP2/TRAP),have been extensively investigated (1, 2). Across pathosystems, proteinsinvolved in host-pathogen interactions are the molecules of choice forvaccine development. Likewise, CS and SSP2/TRAP have become majortargets for intervention and are being actively pursued as vaccinecandidates (3-6). While the results from these trials have beenencouraging, they have revealed that the immunological protectionagainst malaria is not conferred due to a dominant immune responseagainst a single antigen but is mediated by the summation of many modesthumoral and cell-mediated immune responses against a large variety ofknown and unknown antigens (7). Therefore, identification of malarialproteins that are involved in disease pathogenesis will not only lead toa better understanding of the disease process, but is also vital for thedevelopment of a successful vaccine against malaria. With theavailability of the genome sequence and proteome analysis of P.falciparum parasites (8, 9), efforts are now being made to mine thisinformation for identification and characterization of proteins thatcontribute towards pathogenesis (10).

In recent years, the concept of protein domains and domain families hasrisen to greater prominence due to an increasing realization that byorganizing proteins sequences from distinct organisms into domainfamilies, one can often reliably predict their molecular functions (11,12). In case of pathogens, identification of adhesive domain-containingproteins has played a pivotal role in deciphering the mechanics ofdisease pathogenesis. For example, the Plasmodium genome encodes severalproteins that contain an adhesive thrombospondin type I repeat (TSR)domain, most of which have now been shown to be involved inhost-parasite interactions (1, 2, 10, 13). Therefore, identification andcharacterization of parasite proteins containing adhesive domains willimprove our understanding of the disease process and here we describe anovel malarial protein that encodes a single fasciclin 1 (FAS1) domain.

FAS1 is an adhesive domain named due to its initial discovery inproteins involved in fasciculating axons and growth cones (14). It is anancient extracellular adhesive module found in proteins of prokaryotic,plant and animal origin (15-18). Most of the FAS1 domain-containingproteins possess multiple copies of the domain, though proteins encodingonly a single copy, have also been identified (17). A large number ofFAS1 domain containing proteins have been reported in Drosophila andGrasshopper, where they are involved in neuronal development (19, 20).In contrast, in humans, FAS1 domains have been found in a largemulti-domain scavenger receptor protein on endothelial cells, involvedin the removal of hyaluronan from blood stream (21), as well as inextracellular matrix protein, where they mediate corneal epithelial celladhesion (22). However, unlike many domains which show a high degree ofsequence conservation, FAS1 domains show huge sequence diversity;typically have 20% sequence identity in a pairwise alignment (23) andare recognized by only two short semi-conserved sequence motifs(underlined in FIG. 3).

Here we describe a novel P. falciparum FAS1 domain-containing proteinand its role in malaria infectivity during sporozoite stage of thelifecycle. We demonstrate that the protein contributes towards livercell adhesion and invasion by the parasite and have named it asFasciclin Related Adhesive Protein or FRAP.

Materials and Methods

Sequence analysis and identification of FRAP orthologs: Sequences for P.falciparum (Accession #AAN37059), P. berghei (Accession #CAH94515) andP. chaubaudi (Accession #CAH77280) FRAP were obtained from GenBank,where they have been deposited as part of the parasite genome sequencingprojects (8, 24) Using P. falciparum FRAP sequence, orthologs wereidentified from unannotated genome sequences of P. gallinaceum, P.reichenowi, P. vivax, P. yoelii and P. knowlesi parasites, available atPlasmoDB, Sanger Center and TIGR web sites (25). FRAP orthologs fromTheileria parva (Accession #EAN32245) and T. annulata (Accession#CA176887) were from the published genome sequence (26, 27). The nucleicacid sequences of the genes are provided in FIG. 2A-J. The amino acidsequences were aligned using Clustal W algorithm (28) for multiplesequence alignment, using the DNASTAR package. The amino acid sequencesare depicted in FIG. 1, and the alignment is given in FIG. 3.

Reverse Transcription, Amplification and Cloning of FRAP proteins: TotalRNA was obtained from highly purified preparations of P. falciparum (3D7strain) sporozoites (29). 2μg of total RNA was reverse transcribed andamplified with the forward 5′ CACCATGAAAAATAGATTTTATTATAATTTG 3′ (SEQ IDNO: 22) and reverse 5′ AAAAATGATGGGCTTATCTACTATATG 3′ (SEQ ID NO: 23)primers, using Promega Access RT-PCR kit. The amplified fragment wascloned inpET101-TOPO (Invitrogen) an E. coli expression vectorcontaining a C-terminal [His]₆ tag, giving rise to plasmid pFRAP. Theforward primers encoded a tetra nucleotide CACC, which facilitated thedirectional cloning of amplified fragments in the expression vector. Theauthenticity of the clone was verified by DNA sequencing. Two other FRAPconstructs, encoding amino acids 1-87 and 88-205 were generated byPCR-based subcloning using pFRAP as template, giving rise to plasmidpFRAP2 and pFRAP3 respectively. Authenticity of these constructs wasverified by DNA sequencing. Sequencing was performed at the corelaboratory sequencing facility of the Virginia Bioinformatics Institute.

Expression, localization and purification of recombinantly expressedFRAP protein: For protein expression, E. coli BL21 cells weretransformed with a desired plasmid, grown in super broth, and at theOD₆00=1.0, expression was induced with IPTG, at a final concentration of1 mM. Three hours post-induction, the culture was harvested bycentrifugation at 3000 g for 10 minutes. To identify the intracellularsite of accumulation of the protein, pellet was resuspended in 20%sucrose solution in 20 mM Tris pH 7.5 and incubated on ice for 10 min.Cells were spun at 5000 g for 20 min and the pellet was resuspended inchilled water for 10 minutes. This was followed by centrifugation at8000 g for 20 minutes to isolate periplasmic fluid. Spheroplast pelletwas further processed to isolate inclusion bodies, as previouslydescribed (30). Inclusion bodies were solubilized in 1550 mM CAPS buffercontaining 0.3% N-lauryl sarkosine and 0.3 M NaCl, pH 11.0 for 30 minand centrifuged at 10000 g for 30 min at room temperature. Thesupernatant was loaded onto a His-Trap High Performance affinity column(GE Health Care) and bound protein was eluted using an imidazolegradient in 50 mM CAPS pH 11.0 containing 0.3% N-lauryl sarkosine and0.3 M NaCl. Relevant fractions were pooled and purified to homogeneityby gel filtration chromatography on Superdex 200 10/300 GL column (GEHealth Care). Authenticity of the purified protein was verified by aminoterminal sequencing and western blotting using anti-polyhistidine tagmonoclonal antibody. For obtaining recombinant CS protein, pCS271IVC aplasmid with a polyhistidine tag at the carboxyl terminus (1) wasexpressed in BL21 E. coli cells and the protein was purified from theperiplasm as previously described (31).

Generation of anti-FRAP2 antibodies: The protocol for antibodygeneration was approved by the animal care committee at Virginia Tech.6-8 weeks old female CD1 mice were subcutaneously immunized with 10 μgof purified FRAP2 emulsified in complete Freunds adjuvant. Twosubsequent booster doses in incomplete Freunds adjuvant wereadministered on days 21 and 35, after the first immunization. Sera werecollected two weeks after the last booster. Antibodies were purified ona Protein G affinity column using AKTA FPLC chromatography system.

Confocal analysis: Purified P. falciparum sporozoites were air dried ona glass slide. The slide was blocked with 5% normal goat serum inphosphate buffer saline (PBS). Subsequently, the slide was incubatedwith doubling dilutions (1:20 to 1:20480) of anti-FRAP2 or pre immunemouse serum and incubated at room temperature, in a humidified chamber,for one hour. Unbound antibodies were removed by washing the slide withTBS containing 0.05% Tween 20 followed by the addition of an anti-mouseFITC conjugate (1:500 dilution). Confocal imaging was performed usingBioRad Radiance confocal microscope.

Immunoelectron microscopy: Preparations of Plasmodiumfalciparum-infected salivary glands were fixed in 4% paraformaldehyde(Electron Microscopy Sciences, PA) in 0.25 M HEPES (pH 7.4) for 1 hr atroom temperature, then in 8% paraformaldehyde in the same bufferovernight at 4° C. They were infiltrated, frozen and sectioned aspreviously described (32). The sections were immunolabeled with mouseanti-FRAP antibodies (1:1000 in PBS/1% fish skin gelatin), then withanti-mouse IgG antibodies, followed directly by 10 nm protein A-goldparticles (Department of Cell Biology, Medical School, UtrechtUniversity, the Netherlands) before examination with a Philips CM120Electron Microscope (Eindhoven, the Netherlands) under 80 kV.

Liver Cell binding assay: The binding of proteins was assayed on HepG2cells as described previously (1, 31). Briefly, cells were plated at adensity of 25,000 cells/well, in a 96 well plate, 36 hours before thestart of the experiment. The cells were fixed with paraformaldehyde,blocked with 1% BSA, followed by the addition of equimolarconcentrations of recombinant proteins. Bound protein was detected usinganti-polyhistidine tag monoclonal antibody (1:10,000) and anti-mouseantibody conjugated to alkaline phosphatase (1:2000). Amount of boundprotein was detected by using 4-methylumbelliferyl phosphate, afluorescent substrate, and measurement of fluorescence using afluorescent plate reader (Molecular Devices, CA) with excitation andemission set at 350 nm and 460 nm respectively. Results are shown asmean±standard deviation of mean of a representative experiment performedin triplicate. Binding inhibition assays were performed by combining therecombinant proteins with increasing amounts of glycosaminoglycans andincubating at 37° C. for 15 min. For enzyme treatment, cells wereincubated with different concentrations of Heparinase I orChondroitinase ABC for 90 minutes at 37° C. as previously described(31), before the addition of proteins. The bound protein was assayed asdescribed above.

All the proteins used in the binding assay possessed a polyhistidine tagat their carboxyl terminus. Therefore, binding activity was probed usinga polyhistidine tag monoclonal antibody. This excluded the possibilityof misinterpretation of the data due to differences in antibodyaffinities.

Sporozoite Invasion Assay: Invasion assay was performed with HepG2(Human hepatoma) cells as previously described (31). Briefly, HepG2cells were plated (50,000 cells/0.3 ml) and incubated overnight at 37°C. in a CO₂ incubator. Next day, medium was removed and 50 μl of dilutedFRAP proteins (final concentrations: 20 and 10 μg/ml) or anti-FRAP2antibodies (40 μg/ml final concentration) were added per well. Anti CSmonoclonal antibody NFS1 was used at a final concentration of 100 μg/ml.All protein concentrations and serum dilutions were evaluated intriplicate. This was immediately followed by the addition of 20,000sporozoites in 50 μl of medium to each well. P. falciparum (strain NF54)sporozoites were obtained from the salivary glands of An. stephensimosquitoes as described by Ozaki (33). The sporozoites were allowed toinvade liver cells for three hours followed by the washing of cells withPBS at pH 7.4. Subsequently, the cells were fixed with cold methanol.Sporozoites were visualized by immunostaining using NFS1 as primaryantibody and anti-mouse IgG-peroxidase conjugate. The slides weremounted with Paramount and only intracellular sporozoites were countedas described (31). Percentage inhibition of invasion was calculated withthe following formula: [(Control-test)/control]×100

Results

Identification and sequence analysis of FRAP: Analysis of the publishedDNA sequence of chromosome 14 of P. falciparum (8) identified a 824nucleotide sequence (Accession #NP702335) containing a hypothetical,single copy, three-exon gene, encoding a 205 amino acids long protein(FIG. 1, SEQ ID NO: 1). Bioinformatical analysis of the predictedprotein using the NCBI conserved domain database (CDD) search tool (34),revealed that the protein encodes a Fasciclin (FAS1) domain (SMARTaccession no. SM00554) from amino acids 88-204 with an e-value of 2e-10.FIG. 1 depicts the FRAP protein sequence and its alignment with theconsensus sequence of FAS1 domain in the database. FAS1 domains areknown for their huge sequence diversity and typically have 20% sequenceidentity in a pairwise alignment (23). They are recognized by only twoshort semi-conserved sequence motifs (underlined in FIG. 3). A similarpattern is seen in FRAP as its FAS1 domain has 21% sequence identitywith the consensus sequence.

Using published, unpublished and unannotated sequences in the databasesfor pathogens at Sanger, PlasmoDB and TIGR web sites, P. falciparum FRAPorthologs were identified in all Plasmodial species that have beensequenced till date or are currently undergoing sequencing (FIG. 1).Orthologs of P. falciparum FRAP were found in avian (P. gallinaceum),rodent (P. berghei, P. yoelii and P. chaubaudi) simian (P. knowlesi andP. reichenowi) and human (P. vivax) malaria parasites suggesting thatthe FRAP protein is most likely present in all the members of Plasmodiumgenus and, hence, could be playing an important role in the biology ofthe parasite. Within the Plasmodium genus, the protein maintains a 60%sequence identity (FIG. 3) with 124 out of 205 residues being identical.Beyond Plasmodium, FRAP homologs were only found in the two recentlysequenced Theileria genomes (26, 27) with an overall sequence identityof 29% (FIG. 3). In contrast, FRAP homologs could not be found in therecently sequenced Leishmania (35) and Trypanosome genomes (36). Thisselective presence in Plasmodium and Theileria genomes could pointtowards a common function of the protein between otherwise two verydifferent parasites.

The amino acid sequences for the FRAP proteins discussed above aredepicted in FIG. 1, the nucleic acid sequences that encode the proteinsare depicted in FIG. 2, and the corresponding SEQ ID NOS: are given inTable 3. TABLE 3 SEQ ID NOS: for amino acid and nucleic acid SEQ ID NO:Organism Amino acid sequence Nucleic acid sequence P. falciparum SEQ IDNO: 1 SEQ ID NO: 2 P. gallinaceum SEQ ID NO: 3 SEQ ID NO: 4 P.reichenowi SEQ ID NO: 5 SEQ ID NO: 6 P. vivax SEQ ID NO: 7 SEQ ID NO: 8P. yoelii SEQ ID NO: 9 SEQ ID NO: 10 P. knowlesi SEQ ID NO: 11 SEQ IDNO: 12 P. chaubaudi SEQ ID NO: 13 SEQ ID NO: 14 P. berghei SEQ ID NO: 15SEQ ID NO: 16 T. parva SEQ ID NO: 17 SEQ ID NO: 18 T. annulata SEQ IDNO: 19 SEQ ID NO: 20Cloning of P. falciparum FRAP: Coding sequence of P. falciparum FRAP wasamplified by RT-PCR using total RNA from the sporozoite stage of theparasite, giving rise to a 615 bp fragment. This PCR product was not dueto the presence of contaminating genomic DNA in the RNA preparation, asa parallel reaction performed in the absence of reverse transcriptaseenzyme, showed no amplification. Also, the size of the amplifiedfragment, viz. 615 bp, matched the size of the predicted mature mRNA(FIG. 4 b). The amplified fragment from the sporozoite stage was clonedin a T7 promoter-based E. coli expression vector, giving rise to plasmidpFRAP. Sequencing of the cloned DNA fragment authenticated the predictedexon structure and coding sequence for the FRAP protein (data notshown). To investigate the role of FAS1 domain in the biology of theprotein, two more plasmid constructs viz., pFRAP2 and pFRAP3, weregenerated by sub-cloning, using pFRAP as template. pFRAP2 encoded theDNA sequence for amino acids 1-87 of the full length protein whilepFRAP3 encoded the FAS1 domain represented by amino acids 88-205 (FIG. 4a). The authenticity of these clones was also verified by sequencing.Recombinant Expression and Purification of FRAP proteins: To obtainrecombinant FRAP proteins, the desired construct was transformed in E.coli BL21 cells and the expression was induced with IPTG. Three hourspost induction, the culture was harvested and the site of accumulationof the recombinant protein was evaluated by sub-cellular fractionation.For all three FRAP proteins, the expression was localized in thespheroplast in the form of insoluble inclusion bodies (data not shown).Spheroplast pellet was further processed to isolate inclusion bodies, aspreviously described (30). Inclusion bodies were solubilized and theproteins were purified by a combination of affinity and gel filtrationchromatography. The presence of a polyhistidine tag at the carboxylterminus of the recombinantly expressed proteins facilitated thepurification and all three proteins were initially purified on aHis-Trap affinity column (data not shown). The proteins at this stagewere 95% pure. Further purification to apparent homogeneity was done bygel filtration chromatography (FIG. 4 c). Purified FRAP, FRAP2 and FRAP3had the expected molecular weights of 27.8, 12.3 and 17.7 kDarespectively and were recognized by a monoclonal antibody directedagainst the polyhistidine tag present at the carboxyl terminus of allthe proteins (FIG. 4 d). The first 15 residues of each of the proteinswere also verified by amino terminal sequencing (data not shown).Together, these results authenticated the recombinant proteins andsuggested that they were structurally intact.FRAP is localized in the micronemes of the sporozoites: To detect theexpression of FRAP on sporozoites, protein-specific antibodies wereraised by immunizing mice with FRAP2 protein. Anti-FRAP2 antibodiesreadily recognized the expression of FRAP protein on the sporozoite (notshown). The binding was specific as pre-immune serum did not recognizeany expression on the sporozoites. This indicated that transcription ofFRAP mRNA can be correlated to its expression during the sporozoitestage of the lifecycle. Immunoelectron microscopy using anti-FRAP2antibodies revealed that FRAP was localized in the lumen of micronemes,a specialized secretory organelle in the cytoplasm (not shown). Theprotein was present in the apical micronemes, suggesting that it couldbe secreted during the infectivity process. In Plasmodium, micronemescontain several adhesive domain-containing proteins that are associatedwith host cell adhesion and invasion at both, sporozoite anderythrocytic stages of its lifecycle (13, 37, 38). This suggested thatFRAP could be playing a role in the infectivity process.FRAP is involved in adhesion of sporozoites to liver cells: FRAP wasinvestigated for its possible role in host cell adhesion using a humanhepatocyte cell line, HepG2, an established model for investigatingsporozoite-liver cell interactions in malaria (1, 31). FRAP showed adose dependent binding on liver cells (FIG. 5) which was comparable tothe binding activity of CS protein, a known parasite protein involved inthe adhesion and invasion of liver cells by the sporozoites (1). Thissuggested that FRAP could be serving as one of the parasite ligands inhost-parasite interactions. This host-cell binding activity of FRAP wasnot due to the presence of the FAS1 domain alone, as FRAP3, a proteinencoding only the FAS1 domain (amino acids 88-205) did not bind to livercells, even at the highest concentration used in the assay (54).Although FAS 1 domain alone did not show any binding, its deletion fromthe full length protein (protein FRAP2) lead to a 50% loss of activity,in comparison to the full length protein (FIG. 5). This suggested thatboth, FAS1 domain and the amino terminus region, contribute to thebinding activity of the protein and an intact FRAP is required for itsoptimal activity.FRAP binds liver cells through heparan sulfate proteoglycans: As FRAPshowed potent liver cell binding, the nature of its receptor on livercells was investigated by utilizing glycosaminoglycans as competitiveinhibitors. Inhibition of adherence by the addition of solubleglycosaminoglycans in an assay may suggest that the involved hostreceptor is a proteoglycan (31, 39). In the presence of free heparin,binding activity of FRAP and FRAP2 was reduced by 55 and 60%respectively (FIG. 6). In contrast, chondroitin sulfate A showed noinhibition at the highest concentration evaluated in the assay (FIG. 6).This suggested that FRAP utilizes heparan sulfate-based proteoglycans(HSPG) as a receptor for adhesion.

The involvement of HSPG as a receptor was further verified by evaluatingthe binding of the protein on liver cells that were pretreated withspecific glycosaminoglycan-cleaving enzymes. Cells were pre-treated withheparinase I or chondroitinase ABC followed by the evaluation of bindingactivity of FRAP and FRAP2. Heparinase I selectively removes heparansulfate while chondroitinase ABC cleaves chondroitin sulfate A, B and Ctype sugars from the liver cell surface. Both, FRAP and FRAP2 lost 50%of their binding activity on heparinase I treated cells (Table 4)confirming the involvement of a heparin-based receptor on the liver cellsurface. CS protein, which binds hepatocytes through HSPG (39) alsoshowed a similar decrease in binding activity. In contrast, treatment ofliver cells with chondroitinase ABC resulted in no loss of activity.TABLE 4 Binding of FRAP proteins to hepatocytes is inhibited bypretreatment of cells with glycosaminoglycan cleaving enzyme. Cells werepretreated with different concentrations of either Heparinase I orChondroitinase ABC for 90 minutes followed by the addition of 100 nM ofprotein. Inhibition of binding was calculated by comparing the bindingof respective proteins on non-treated HepG2 cells in the same plate.Inhibition of Binding (%) Enzyme, U/ml FRAP FRAP2 CSP Heparinase I 1.2539.4 ± 4.2  42.3 ± 10.4  48.1 ± 12.0 2.50 42.1 ± 7.6 41.4 ± 1.5 57.7 ±7.9 5.00 47.8 ± 1.4 49.4 ± 9.2 59.1 ± 6.5 Chondroitinase ABC 0.01 — — —0.12 — — — 1.25 — — —

FRAP is involved in liver cell invasion: As FRAP proteins efficientlybound to HepG2 cells, we investigated the ability of the two proteinsand the anti-FRAP2 antibodies in preventing invasion of human livercells by P. falciparum sporozoites in culture. Both FRAP and FRAP2 couldprevent sporozoites from invading liver cells by 89.5% and 92.4%respectively, at the highest concentration of the protein used in theassay. This activity was comparable to the invasion inhibition activityof CSP protein, which at a similar concentration could also inhibit theinvasion by 92.6%. Anti-FRAP2 antibodies showed extreme potency as at aconcentration of 40 μg/ml, it inhibited sporozoite invasion by 94.6%, alevel comparable to the inhibitory activity of anti-CS monoclonalantibody NFS1 (Table 5). This indicated that (i) FRAP not only plays arole in binding, it is also involved in the invasion process (ii) theprotein utilizes its amino terminus (amino acids 1-87) for its invasionactivity and (iii) a potent antibody response against FRAP2 by the hostmay play a role in malaria control. TABLE 5 FRAP is involved in invasionof liver cells by P. falciparum sporozoites. Invasion of HepG2 cells byP. falciparum sporozoites was evaluated in the presence of differentconcentrations of free proteins or anti-FRAP2 antibodies and comparedwith the invasion activity in the presence of culture medium. %inhibition represents the decrease in the number of sporozoites thatinvaded liver cells in comparision to the invasion level in cellsincubated with culture medium. Concentration Treatment μg/ml %Inhibition Culture Medium — FRAP 20 89.5 + 1.0 10 80.9 + 1.0 FRAP2 2092.4 + 3.5 10 88.1 + 4.6 CS Protein 20 92.6 + 2.0 Anti-FRAP2 antibody 4094.6 + 1.2 Anti-CS monoclonal 100 97.4 + 0.7Discussion

Deciphering the mechanism of infectivity of the malaria parasite is amajor prerequisite for developing intervention strategies. Key to thisprocess is the unique set of proteins, many of them currently unknown,expressed by the parasite to bind and invade host cells. Therefore, acombination of biochemical and functional studies of malarial genes isrequired to identify parasitic proteins involved in pathogenesis.

We identified P. falciparum FRAP, a new parasite protein and showed thatit is expressed during the sporozoite stage of the lifecycle. Orthologsof P. falciparum FRAP were identified in rodent, avian, simian and humanmalaria species and multiple sequence alignment revealed that theprotein has 60% sequence identity within the Plasmodium genus (FIG. 3).Its universal presence and conserved nature suggested that the proteinplays an important role in the biology of the parasite.

The protein was localized in the sporozoite micronemes by immunoelectronmicroscopy. Micronemes are specialized secretory organelles inPlasmodium and during the sporozoite stage secrete a wide variety ofproteins involved in parasite motility, traversal and host cellinfection. Previously, TRAP/SSP2 and SPECT, two sporozoite proteins withadhesive Thrombospondin type I repeat (TSR) domains have been found inthe micronemes and have subsequently been shown to be involved in theinfectivity process (13, 37). As FRAP encoded FAS1, an ancient adhesivedomain present in both prokaryotes and eukaryotes, we thereforeinvestigated the role of FRAP in host cell adhesion and invasion by thesporozoites.

The protein was recombinantly expressed in E. coli and purified tohomogeneity by column chromatography (FIG. 4 c). The purified proteinshowed robust and dose dependent binding to liver cells indicating thatit is involved in the attachment of sporozoites to liver cells (FIG. 5).This activity was comparable to the binding activity of CS protein,considered to be the primary binding ligand, suggesting that FRAP couldbe one of the primary parasite proteins involved in attachment ofsporozoites to liver cells. In βig-h3, a FAS1 domain-containing humanprotein involved in corneal cell adhesion, the adhesion activities ofthe protein completely resides in the FAS1 domain (22). To investigatethe role of FAS1 domain in FRAP, we expressed FAS1 domain alone (aminoacids 88-205, protein FRAP3) and evaluated its cell binding activity onHepG2 cells. The protein did not show any cell binding activity (FIG.5), indicating that the deleted segment (amino acids 1-87) of theprotein plays an important role in the binding activity of the protein.

This was investigated by expressing amino acids 1-87 (protein FRAP2) inE. coli and evaluation of its cell binding activities on the liver cellline. FRAP2 was capable of binding to liver cells, albeit at only halfthe strength of its full length protein, FRAP. This suggested that aminoterminus region of the protein plays an important role in the host cellbinding, however, an intact FRAP molecule is required for its optimalactivity. The loss of activity seen here could be due to loss of therequired tertiary conformation of the binding domain (due to the absenceof the FAS1 domain) and/or part of the binding motif is present in theFAS1 domain of the protein. A similar situation exists in the case of CSprotein, where the unique amino terminus region plays an important rolein liver cell binding and invasion activities of the protein (31).

FRAP exploited heparan sulfate proteoglycans, expressed on liver cellsurface, as receptor for its biological activities (Table 4). This wasrevealed by competition studies with defined carbohydrates, as well asloss of binding upon enzymatic removal of host glycans. Heparansulfate-protein interactions involve positively charged residues of theprotein, which interact with the negatively charged carboxylate andsulfate ions of the glycosaminoglycan chain. The amino terminus of FRAPpossesses a disproportionate number of positively charged residues (13out of the first 50) some of which are extremely conserved within thePlasmodium genus (FIG. 3). Their conserved nature suggests that theycould possibly be involved in these interactions. Parallels exist forsuch mechanism in other heparin-binding proteins where a large number ofpositively charged residues involved in heparin/HS interaction arepresent in a close proximity in the protein (40).

Entry of sporozoites into the hepatocyte is a multistep process, wherethe initial attachment to the hepatocytes is followed by the invasion ofliver cells, by the parasite. To investigate the role of FRAP in theinvasion process, recombinant FRAP proteins and anti-FRAP2 antibodieswere used as competitors in an in vitro invasion assay. Proteins FRAP,FRAP2 and anti-FRAP2 antibodies inhibited the invasion of liver cells byP. falciparum sporozoites with extreme competence, showing as high as94.6% inhibition (Table 5) in the assay. These levels were comparable tothe inhibitory activity of CSP protein and anti-CSP monoclonal antibody.These results indicated that FRAP is utilized by sporozoites for bothadhesion and subsequent invasion of liver cells and the amino terminalregion plays an important role in these processes. It is noteworthy thatsimilar level (>90%) of inhibition has only been possible by targetingCSP, SSP2/TRAP and the recently discovered SPATR protein (10). Recently,AMA1 has been shown to be involved in liver cell invasion but antibodiesagainst the protein could inhibit the invasion only by about 50% (41).CSP and SSP2/TRAP are being vigorously pursued as vaccine candidates andare currently being evaluated in the clinic (4, 5). Involvement of FRAPin liver cell invasion and its strong inhibition by antibodies suggestthat a potent immunological response against this protein in vivo couldserve as a strategy for intervention and the immunological competence ofFRAP as a vaccine candidate needs to be investigated.

Although we have investigated the role of FRAP in the liver celladhesion and invasion by the sporozoites, it is noteworthy thatmicroarray and proteomic studies have revealed that FRAP is alsotranscribed and expressed during the erythrocytic stages of thelifecycle, especially during the schizonts, which is immediatelyfollowed by the release of merozoites and invasion of red blood cells(9, 42, 43). AMA1 and MAEBL, two micronemal proteins that are expressedat sporozoites and erythrocytic stages of the lifecycle, are involved inpathogenesis, both, at pre-erythrocytic and blood stages, where theyplay a role in host cell adhesion and invasion (41, 44-46). With itsmultistage expression, it is possible that FRAP could also be involvedin host-parasite interactions during erythrocytic stages of thelifecycle.

In conclusion, we have identified and characterized a new parasiteprotein involved in malaria pathogenesis at the sporozoite stage of thelifecycle. It's involvement in pathogenesis indicates that developingintervention strategies targeting FRAP creates new treatment options forcontrolling malaria.

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Example 2 Inhibitory Epitope in FRAP

Identification of Inhibitory epitope by peptide mapping As we havedemonstrated that antibodies against FRAP2, an 87 amino acid polypeptidecan prevent invasion, the region of the protein responsible for thisrecognition was mapped by developing a set of overlapping peptides thatwere utilized for ELISA. A set of 10 overlapping peptides (Table 6) werechemically synthesized and used as coating antigen to identify theepitope recognized by these antibodies. Overlapping Peptides HAI-3,4, &5 were predominantly recognized by these antibodies, suggesting that a32 amino acid sequence (TRSGGLRKPQKVTNDPESINRKVYWCFEHKPV, SEQ ID NO:24), comprised by these peptides is being recognized by the inhibitoryantibodies (Table 7). A sequence comparison of these peptides revealsthat an 8 amino acid sequence (TNDPESIN, SEQ ID NO: 37) is present inall of them (FIG. 7) suggesting that this sequence could be an importantcomponent of the region recognized by the anti-protein antibodies.Therefore, the 32 amino acid sequence or portion thereof can beexploited as part of a multi-epitope subunit vaccine. The 32 amino acidregion has 100% sequence homology or 87.5% sequence identity within thePlasmodium genus implying that this region plays a critical role in allthe Plasmodium species and an immune response(s) generated against thisregion of the protein in one species could be a factual representationof immnune responses against other species, generated by its host. Twoother peptides (HAI-7 and HAI-10) were also recognized by theanti-protein antibodies suggesting that their recognition is alsoimportant in preventing parasites from initiating an infection. TABLE 6Sequence of peptides chemically synthesized for identification ofinhibitory epitope. Peptide Sequence SEQ ID NO HAI-1MKNRFYYNLIIKRLYTRSGG SEQ ID NO: 27 HAI-2 NLIIKRLYTRSGGLRKPQKV SEQ ID NO:28 HAI-3 TRSGGLRKPQKVTNDPESIN SEQ ID NO: 29 HAI-4 GLRKPQKVTNDPESINRKVYSEQ ID NO: 30 HAI-5 TNDPESINRKVYWCFEHKPV SEQ ID NO: 31 HAI-6VYWCFEHKPVKRTIINLIYS SEQ ID NO: 32 HAI-7 KPVKRTIINLIYSHNELKIF SEQ ID NO:33 HAI-8 NLIYSHNELKIFSNLLNHPT SEQ ID NO: 34 HAI-9 NELKIFSNLLNHPTVGSSLISEQ ID NO: 35 HAI-10 NLLNHPTVGSSLIHELSLDG SEQ ID NO: 36

TABLE 7 Recognition of FRAP-derived peptides by ELISA. Peptides werecoated onto the ELISA plate followed by the addition of log dilutions ofantibodies followed by anti-mouse antibodies conjugated to alkalinephosphatase. Recognition was measured at 405 nm using an ELISA platereader. Peptide/ 1:100 1:1000 1:10K Antigen FRAP FRAP2 FRAP FRAP2 FRAPFRAP2 HAI-1 0.010 0.015 — — — — HAI-2 0.010 0.020 — — — — HAI-3 0.7100.530 0.223 0.190 0.026 0.020 HAI-4 0.789 0.710  0.4445 0.630 0.0630.230 HAI-5 0.660 0.636 0.290 0.465 0.030 0.110 HAI-6 0.005 — — — — —HAI-7 0.730 0.065 0.550 0.026 0.165 — HAI-8 0.020 0.290 — 0.039 — —HAI-9 0.030 — — — — — HAI-10 0.250 0.300 0.030 0.045 — — FRAP* 0.6700.600 0.650 0.465 0.340 0.130 FRAP** 0.210 0.260 0.070 0.400 — 0.190*4 pmol of protein**2 nmol of each peptide in 50 ul coating buffer

Optimal recognition of an epitope by the host immune system requiresthat the epitope maintains its structural conformation. While shortamino acid sequences can be easily recognized in vitro, theirrecognition under in-vivo conditions almost always requires them to bepresent as part of a much larger polypeptide. This is especiallyimportant for configurational epitopes present in the surface antigensof malaria parasite whose recognition requires that a continuous stretchof amino acids, larger than its identified epitope, be present for itsoptimal recognition. Therefore, a 32 amino acid long region is mostlikely required for optimal recognition of FRAP protein by the hostimmune system and it could be utilized either alone or in combinationwith other known and unknown malarial antigens in a vaccine.

-   FRAP is recognized by the host immune system of malaria-infected    subjects. Sera from 17 malaria infected subjects was screened for    the presence of anti-FRAP antibodies, by ELISA.

0.5 microgram of purified FRAP protein was coated as antigen and itsrecognition was probed with sera at 1:200 dilution. 4 sera samples fromnorth American volunteers, who have never been exposed to malaria wereused as control. A cutoff value of OD405=0.378, which represented meanof OD+2 SD was used to determine samples that were positive. The ELISAresults indicated that 10 out of 17 (58.8%) infected subjects hadanti-FRAP antibodies (Table 8) with OD values above the set cutoff TABLE8 Recognition of full length FRAP by sera from infected subjects livingin Bandiagara, a malaria-endemic district in Mali. Sample ID Absorbance,405 nm Positive 1A-001 0.79 ± 0.07 Y 1A-002 1.03 ± 0.05 Y 1A-004 0.47 ±0.02 Y 1A-005 0.23 ± 0.01 N 1A-007 0.23 ± 0.01 N 1A-008 0.49 ± 0.07 Y1A-010 0.26 ± 0.01 N 1A-011 0.21 ± 0.01 N 1A-013 0.91 ± 0.00 Y 1A-0140.30 ± 0.01 N 1A-016 0.60 ± 0.01 Y 1A-017 0.15 ± 0.01 N 1A-019 0.43 ±0.00 Y 1A-020 0.56 ± 0.01 Y 1A-021 0.40 ± 0.00 Y 1A-023 0.41 ± 0.02 Y1A-024 0.20 ± 0.00 N

Example 3 FRAP is a Malaria Drug Target

Once a malaria parasite infects red blood cells, host hemoglobin servesas its primary source of amino acids required for its geometric increasein infection. It achieves its goal by cannibalizing hemoglobin to itsconstituent amino acids, which it recycles for its own proteinsynthesis. While the parasite is extremely effective in digesting theprotein (globin) component of hemoglobin the heme prosthetic groupserves as a challenge to its survivability. Free heme released fromhemoglobin is lethal for the parasite and to escape its deleteriouseffects the parasite enzymatically polymerizes heme into a non-toxicbyproduct known as hemozoin. Therefore, any mechanism by whichpolymerization of heme into nontoxic hemozoin can be inhibited will leadto a very effective therapeutic for malaria.

We show here that FRAP is responsible for this activity. FRAPeffectively converted toxic heme into inactive hemozoin in a dosedependent manner (FIG. 8). The hemozoin formation activity was10-20-fold higher in comparison to histidine rich protein II, the onlyknown parasite protein capable of making hemozoin. This activity wasspecific as it was lost when the protein was pre-treated with proteinaseK (a non specific protease) suggesting that an intact protein isrequired for this activity (FIG. 9). The activity requires the completeprotein as two truncated variants of FRAP (FRAP2 and FRAP3) did not showany hemozoin formation (FIG. 8).

The authenticity of the polymerized heme as hemozoin was verified byFT-IR spectroscopy. The IR spectra of hemozoin contains an intenseabsorbance at 1664 and 1211 cm⁻¹, that are absent in the spectra of freeheme (Slater et al., 1991). These are characteristics of a carboxylategroup coordinated to the iron center of ferriporphyrin (Fe01-O41)arising from stretching of the localized carbon-oxygen double and singlebonds, respectively (Slater et al., 1991). The chemical structure ofβ-hematin is depicted in FIG. 10 (adapted from (Pagola et al., 2000)).The infra red spectra of the FRAP-generated product showed thecharacteristic decrease in transmittance at 1664 and 1211 cm⁻¹,chemically validating that the product formed was indeed hemozoin (FIG.11).

FRAP residues involved in heme polymerization were identified bygenerating 11 variants of FRAP by site-directed mutagenesis. Evaluationof these mutants for heme polymerization-activity revealed that threeresidues viz., F42, H44 & H122 are critically involved in hemozoinformation, as their conversion to alanine lead to a complete loss ofactivity (Table 9).

FRAP protein shows remarkably high amount of sequence homology betweendifferent Plasmodium species. In FRAP, a highly conserved proteinsequence has biological relevance as the residues shown to be involvedin hemozoin formation viz., F42, H44, H122 (Table 9) are not onlyconserved within the Plasmodium genus, they are also conserved inTheileria parasites. This indicates that FRAP protein from a non-humanmalaria parasite can be used as target for screening and development ofnovel inhibitors for FRAP protein of human malaria parasite.

This can be achieved by screening a library of smallmolecules/inhibitors in vitro in the FRAP-mediated hemozoin formationassay, which will lead to the identification of a candidate molecule(s).These molecules can be subsequently evaluated in an in vitro P.falciparum culture in the laboratory. Once their efficacy has beenproved in vitro, these molecules can be evaluated in a rodent malariaparasite model. This will be feasible due to the extremely conservednature of the protein and the amino acids residues of FRAP involved inthe process of hemozoin formation (F42, H44, H122), as seen bysite-directed mutagenesis, being identical between all known FRAPproteins (Table 9, FIG. 3).

Once a small molecule shows efficacy in the mouse malaria model, it canbe directly evaluated in a monkey model without requiring extensiveexperimentation as FRAP in P. knowlesi, the monkey malaria parasite, hasthe same residues in its active site. Therefore, it is possible todevelop FRAP inhibitors for human malaria parasite by targeting FRAPsequence from other species of Plasmodium. TABLE 9 Identification ofFRAP residues involved in Hemozoin formation. 11 FRAP residues wereindividually mutated to alanine by site-directed mutagenesis; proteinswere expressed in E. coli and purified to homogeneity. Polymerization ofheme was investigated with 500 pmoles of each of the proteins and theiractivity was compared with the unmutated FRAP. Conversion of F42, H44and H122 lead to a complete loss of activity, suggesting a critical rolefor these residues in the polymerase activity of the protein. HemePolymerized Protein (nmoles) % Decrease FRAP 139.2 — Y39A 155.2 — F42A0.6 99.5 H44A 6.7 95.1 F64A 102.2 26.6 H79A 133.5 4.1 F90A 111.1 20.1H122A 0.9 99.3 C191A 104.9 24.6 H192A 115.6 16.9 H197A 106.4 23.5

A time kinetic analysis for hemozoin formation revealed that theconversion of heme into hemozoin was complete within 5 hours and was pHdependent where a pH of 5.2 was required for optimal activity (FIG. 12).Stoichiometric analysis for FRAP-Heme interaction using continuousvariation method (Job's Plot) revealed that the protein has a 1:1stoichiometry with heme (FIG. 13). Hemozoin formation could beeffectively inhibited by chloroquine, an antimalarial that is known toexerts its activity by binding to free heme and preventing itspolymerization into hemozoin (FIG. 14).

These results clearly demonstrate that (i) FRAP is responsible forneutralization of heme through a polymerase activity and (ii) thepolymerization can be inhibited by chloroquine. In addition, the activesite residues that are critical for this activity were identified.Therefore, FRAP is an efficient drug target for malaria drugdevelopment, for example, for the design of small molecules that bind tothe active site and inhibit the catalytic capability of FRAP.

References for Example 3

-   1. Francis, S. E., Sullivan, D. J., Jr. and Goldberg, D. E. (1997)    Annu Rev Microbiol, 51, 97-123.-   2. Gluzman, I. Y., Francis, S. E., Oksman, A., Smith, C. E.,    Duffin, K. L. and Goldberg, D. E. (1994) J Clin Invest, 93,    1602-1608.-   3. Pagola, S., Stephens, P. W., Bohle, D. S., Kosar, A. D. and    Madsen, S. K. (2000) Nature, 404, 307-310.-   4. Slater, A. F. and Cerami, A. (1992) Nature, 355, 167-169.-   5. Slater, A. F., Swiggard, W. J., Orton, B. R., Flitter, W. D.,    Goldberg, D. E., Cerami, A. and Henderson, G. B. (1991) Proc Natl    AcadSci USA, 88,325-329.-   6. Sullivan, D. J., Jr., Gluzman, I. Y. and Goldberg, D. E. (1996)    Plasmodium hemozoin formation mediated by histidine-rich proteins.    Science, 271, 219-222.-   7. Wellems, T. E., Walker-Jonah, A. and Panton, L. J. (1991) Proc    Natl Acad Sci USA, 88, 3382-3386.

Example 4 Use of FRAP in High Through Put Assays for Hemozoin Formationfor Screening Novel Antimalarials

As described above, the pathway for conversion of heme to hemozoin is amajor drug target. Until now, in vitro screening of small moleculescapable of this blockage has been performed by evaluating their activityin an assay of hemozoin formation, where polymerization is beingperformed using parasite lysate or is chemically driven requiringextremely high salt concentrations. These conditions, though yieldinghemozoin, are far from perfect as a typical experiment requires a 16hour reaction and less than 10% of the substrate is converted into aproduct (Tripathi et al., 2004). Our FRAP-based methodology of hemozoinformation is extremely superior to the currently available technology,as it mimics the in vivo process, converts >50% of the initial substrateinto product and can be completed in as little as 5 hours. Therefore, aFRAP-based assay system for the identification of antimalarials is anassay system of choice for these processes.

Screening Procedure for Inhibitors of FRAP-mediated Hemozoin Formation.

The first assay describes in detail how the hemozoin formation isinvestigated. This is the complete detail of the assay documenting everystep of the process. This assay will be used for studying the role of aninhibitor, as inhibition of FRAP activity will cause a decrease inhemozoin formation which will b easily quantifiable by this assay. Thisassay was used to inhibit hemozoin formation using chloroquine and hasbeen described as assay 2.

Assay 1: FRAP-mediated Hemozoin formation Assay (all temperatures indegree C.):

The standard assay contained in a total volume of 1.0 ml: 500 mM sodiumacetate pH 5.2, 300 nmol/ml hemin-Cl (as substrate) and 500 pmol/mlFRAP, as the source of heme polymerase activity. The amount of FRAPadded was chosen such that 50% of the substrate was converted intoproduct (insoluble hemozoin) during the assay. The reaction wasinitiated by protein addition and allowed to proceed for 16 hours at 37degree. The reaction was terminated by adding 0.01 ml of 10% SDSsolution. The reaction tube was centrifuged at 13,000 rpm for 15 minutesat 23 degrees and the supernatant was carefully removed. The pellet,which contained the polymerized and insoluble hemozoin, was resuspendedin 1 ml of 0.1M sodium bicarbonate pH 9.1 containing 2.5% SDS. At thisstep, any free heme present in the pellet will go into the solution atit is soluble in sodium bicarbonate while the hemozoin is insoluble.This process essentially removes any free heme that could be present inthe pellet. The suspension was spun at 13,000 rpm and the supernatant,containing unpolymerized substrate was removed. This process wasrepeated thrice, followed by washing of the pellet in pure water. Thepellet obtained after final washing was dissolved in 0.3 ml of 0.1N NaOHand the absorbance of the solution was measured at 405 nm using aspectrophotometer. Amount of heme polymerized was calculated utilizing astandard curve, prepared by dissolving known amounts of commerciallyavailable beta-hematin in 0.1N NaOH. Chemically synthesized beta-hematinand biologically polymerized hemozoin are chemically identical (Pagolaet al, 2000 Nature).

To assure that the heme polymerized was due specifically to the actionof FRAP, a parallel control incubations were performed which either didnot contain any protein or contained bovine serum albumin, which wasused a non-specific protein control. Furthermore, the hemozoin formationwas also evaluated with truncated variants and point mutants of FRAP tonot only describe its structural requirements, but also pin point theresidues that are involved in the polymerization process.

Assay 2: Inhibition of FRAP-mediated hemozoin formation

For inhibition studies, the inhibitor under examination was added to thestandard assay cocktail (as described above) at the desiredconcentration and the FRAP-mediated hemozoin formation activity wascompared to that found in control (minus inhibitor) incubations whichlacked inhibitor.

This assay system will be utilized for screening FRAP inhibitors. Adifference in the amount of hemozoin seen in the presence of aninhibitor with respect to the reaction where the inhibitor was absent isdirectly attributable to the activity of the inhibitor in the reaction.

Example 5 siRNA Mediated Inhibition of FRAP Activities and GeneticMechanisms that can Downregulate FRAP Expression Leading to MalariaControl

Gene knockout experiments were performed for FRAP to study itscriticality in the life of the parasite. DNA encoding a short segment ofFRAP was cloned into a vector encoding the gene for Dihydrofolatereductase (DHFR) as a selection marker. The resulting plasmid vector wastransfected into parasites in culture, and the parasites were thensubjected to drug pressure (e.g. Drug WR99210) to select for parasitesthat do not encode a functional FRAP gene. Deletion of FRAP from thegenome led to the death of the parasites indicating that (i) this geneis critical for the survival of the parasite and (ii) any strategy thatcan either prevent the expression of the FRAP gene product or decreaseits level of expression can be exploited for controlling malaria. Thisresult also gains credence from the biological role of this proteindescribed by inventors where they have shown that the protein isinvolved in the infectivity process and in neutralization of heme, whichis critical for the survival of the parasite. Therefore, methods thatcan neutralize the FRAP gene product will automatically lead to malariacontrol.

In the last few years, inhibition of a gene function by utilizing smallinhibitory RNA (siRNA) has been shown to be feasible for a variety ofpathogens. This technology has proved to be extremely effective inTrypanosome parasites, where it has been extensively utilized forunderstanding the role of a particular gene in the infectivity processand pathogenicity (Best et al., 2005; Ullu et al., 2002). As deletion ofFRAP from the genome is lethal, and the protein plays an important rolein the disease process, therefore, siRNA mediated gene silencing can bean effective method for controlling malaria. This is achieved bydesigning short segments of sense and anti-sense RNA fragments that arecomplementary to the coding sequence of FRAP. These sequences aredelivered to the cytosol of the parasite through a plasmid DNAconstruct. Once in the cytosol, transcription of the siRNA occurs andprevents the expression of FRAP. The result is loss of the activity ofthis critical protein, without which the parasite is not able tosurvive.

For example, human Plasmodium parasites can be transformed with vectorsexpressing one or more siRNA molecules based on SEQ ID NOS: 2 or 8.Methods for design of siRNA molecules have been published by a number ofsources. A recent publication by Dharmacon Inc. (Reynolds, A. et al.,Rational design for RNA interference (2004), Nature Biotechnology 22:326-330) suggests eight design criteria optimal for effective siRNAdesign. The siDESIGN™ Center Program provided by Dharmacon Inc. can beused to design optimal siRNA molecules based on the SEQIDs 2 or 8, thathave one or more of the following features: have low G/C nucleotidecontent (30-52% G/C); three or more A/U nucleotides at the 3′-terminusof the sense strand (the mRNA coding strand); a lack of internal repeatsthat can form secondary structures; and sequence-specific preferences atthe following positions on the sense strand—an A at position 19, an A atposition 3, a U at position 10, and an absence of a G or C at position19 and a G at position 13. The resulting siRNA oligonucleotides can becloned as a small hairpin RNAs (shRNA) between a Plasmodium RNAPolymerase III (Pol III) promoter, which initiates synthesis at adefined distance from the promoter, and a termination sequenceconsisting of a string of 4-5 uridines, or other suitable constitutivepromoters can be used as well. When transfected and co-expressed with aselectable marker into Plasmodium cells, siRNA expression will reducethe levels of the endogenous mRNAs corresponding to SEQ ID 2 or 8.

Several sources are available which give detailed descriptions of theuse of siRNA technology. For example, WO0044895 (Kreutzer and Limmer)specifically covers the use of small dsRNAs as therapeutics, andspecifically to methods and medicaments involving the use of smalldsRNAs formed from two separate strands and having a regioncomplementary to the target gene.

US2005026278 (Tuischl et al.) describes a key structural feature ofsiRNAs, namely the presence of overhangs at the 3′-end of each of thetwo strands and includes data on mammalian cell gene silencing. U.S.Pat. Nos. 5,898,031 and 6,107,094 (the entire contents of which arehereby incorporated by reference) describe degradation of target mRNAmediated by chemically modified RNAi-like oligonucleotides.

Example 6 A Variant of FRAP that Leads to Attenuated Parasites, whichcan be Used as a Whole Organism Vaccine

We have successfully demonstrated that FRAP performs the criticalneutralization of toxic heme into non-toxic hemozoin. We have alsoidentified amino acids in FRAP, whose conversion result in proteinvariants in which the heme polymerase activity has been totally lost orhas been compromised (Table 7). Developing a parasite which has beengenetically modified in such a way, where the FRAP gene is present inthe genome, but it has been modified by a genetic modification to avariant copy of the protein, which encodes a protein that is not fullyfunctional, will give rise to attenuated parasites. Such a process hasbeen previously demonstrated in other systems where CSP, a gene encodinga parasite protein involved in pathogenesis was swapped by geneticmanipulation resulting in attenuated parasites (Tewari et al., 2005).Attenuated parasites may also be produced using siRNA vectors asdescribed in the section above.

Example 7 Use of FRAP as a Tool for High Expression of RecombinantProteins and Subsequent Purification

As described in Example 1, expression of DNA encoding FRAP in E. Colileads to very high expression and up to 40 mg of purified protein can bepurified from a one liter shaker flask culture. Obtaining high yieldsfor a recombinant protein and development of optimal purificationstrategies has long been recognized as a major bottleneck for developingtherapeutics. In the field of recombinant protein expression andpurification, these issues have been tackled by expressing a gene ofinterest fused with a second gene (commonly called as a tag), which hasdistinct binding properties and a high level of expression. The two mostcommonly utilized tags for such purposes are DNA encoding formaltose-binding protein and glutathione S transferase. These tags notonly facilitate purification of protein by exploiting the distinctbinding properties of the tags but also help by enhancing the expressionof the gene of interest.

The high level of expression of FRAP in its recombinant expression andits unique capabilities of interaction with heme makes this proteinuniquely fitted to serve as a tag in recombinant expression vectors.FRAP-based fusions proteins are purified by affinity chromatography byexploiting its heme-binding properties in a column chromatographysystem, where the fusion protein binds to the column through availableheme moiety and is cluted by excess of free heme. Various fusionproteins of FRAP having epitopes of CSP and TRAP may be produced by thismethod for use, e.g. in a vaccine.

References for Examples 4-7

-   Best, A., Handoko, L., Schluter, E. and Goringer, H. U. (2005) J    Biol Chem, 280, 20573-20579.-   Tewari, R., Rathore, D. and Crisanti, A. (2005) Cell Microbiol, 7,    699-707.-   Tripathi, A. K., Khan, S. I., Walker, L. A. and    Tekwani, B. L. (2004) Anal Biochem, 325, 85-91.-   Ullu, E., Djikeng, A., Shi, H. and Tschudi, C. (2002) RNA    interference: advances and questions. Philos Trans R Soc Lond B Biol    Sci, 357, 65-70.

Example 8 Production of Fully Human Antibodies

Fully human monoclonal antibodies against Plasmodium or Theileriaantigens are made in mice directly, when these mice are engineered toproduce only human antibody chains. For example the technology practicedby companies such as Abgenix Inc. [XenoMouse technology, U.S. Pat. No.6,657,103], Medarex Inc. and GenMab A/S [HuMab Mouse or UltiMABtechnology; WO2005023177] can be used. Purified proteins as describedabove are used to immunize such engineered mice. Monoclonals produced inthis manner are produced, screened and characterized in the standardmanner. Fully human antibodies are produced using phage display methodsby screening against human antibody phage display libraries. For exampletechnologies practiced by companies such as Cambridge AntibodyTechnology [U.S. Pat. No. 5,969,108 and U.S. Pat. No. 6,172,197] andothers, can be used to identify fully human antibodies in this manner.Phage display screening has as an added advantage that the process doesnot rely on animal immunization. The genes for fully human antibodiesproduced using engineered mice, or identified through phage display, areisolated, sequenced and cloned for expression in mammalian cell linesfor high level expression using standard methods.

Example 9 Further Characterization of HDP

Development of new drugs is urgently needed to replace majorantimalarials that have become ineffective due to increasing drugresistance. During the intra-erythrocytic stage, malaria parasitesproteolyse globin chains of host hemoglobin1, releasing prosthetic groupheme, which is toxic to the parasite. Heme is immediately detoxified,primarily by its conversion into a metabolically inert crystallinematerial called hemozoin (Hz)^(2.3), a step essential for parasitesurvival and targeted by some of the most effective antimalarial drugsever discovered, including chloroquine. These drugs exert theiranti-parasite activity by binding to free heme^(4.5), which prevent itsdetoxification into Hz. Parasite factors responsible for hemedetoxification are poorly identified and remain controversial^(6.9). Inthis example, the identification, genetic characterization andfunctional activity of a novel Plasmodium falciparum protein thatefficiently converts free heme into Hz is described. The protein readilyconverts up to 50% of free heme into Hz, at a rate that is at least anorder of magnitude higher than any of the known parasite factors^(6.9)capable of Hz synthesis. Therefore, the polypeptide has been designatedheme detoxification protein or HDP. (Alternatively, the protein may alsobe designated “Fasciclin Related Adhesive Protein” or “FRAP”, as is thecase in the previous examples. HDP orthologs have also been identifiedin rodent, simian and avian Plasmodium species. HDP is highly conservedwithin the Plasmodium genus and appears to be essential as it's genedisruption could not be achieved in P. falciparum parasites. Byimmunoelectron microscopy studies, it has ben demonstrated that aftermerozoite invasion, ring form parasites express and secrete this proteininto the erythrocyte cytosol before any detectable amount of Hz isvisible inside the parasite. Subsequently, HDP, accompanied by hosthemoglobin, is delivered to the parasite food vacuole, the site of Hzformation. Together, these results establish HDP as a key parasiteprotein responsible for heme detoxification and therefore, its targetingcould lead to the discovery of novel antimalarial drugs.

Major clinical manifestations of malaria are associated with thedevelopment of Plasmodium parasites inside host erythrocytes. Duringthis stage, heme is detoxified and predominantly sequestered inside theparasite's food vacuole as Hz, which is chemically and structurallyidentical to β-hematin^(2.3). The underlying mechanism, though poorlyunderstood, is believed to be highly conserved as Hz formation occurs inall the species of Plasmodium during their intraerythrocyticdevelopment, irrespective of the host species they infect.

HDP, a single copy, three-exon encoded¹⁰, 205 amino acid long P.falciparum polypeptide (GenBank Acc#NP_(—)702335; FIG. 3) that potentlydetoxifies heme into Hz (FIG. 4 c and 4 d) was identified. The HDP genewas found to be actively transcribed and expressed during theintraerythrocytic stages, a phase of the lifecycle where Hz is producedby the parasite (FIG. 4 b). The coding sequence of HDP corresponding toamino acid 1-205 (SEQ ID NO: 1) was cloned in a T7 promoter-based E.coli expression plasmid and recombinant HDP was produced and purified tohomogeneity (FIG. 4 a).

In a Hz formation assay⁶, where heme was present in several hundred foldmolar excess with respect to HDP, it was found that the protein activelyconverted heme into Hz, in a dose dependent manner (FIG. 16 a-b). Hzproduction increased with an increase in the concentration of eitherfree heme (FIG. 16 a) or HDP (FIG. 16 b), converting up to 50% of freeheme into Hz, until the reaction reached equilibrium (FIG. 16 a). At thehighest heme concentration tested, HDP produced Hz at a rate of 21nmol/hr, which was at least 20 fold higher than that of Histidine RichProtein II (HRP II) and unsaturated (oleic acid and mono-oleoylglycerol) lipids (FIG. 16 a), the only known parasite components capableof Hz synthesis. This process was HDP-dependent, as in its absence, Hzproduction occurred at baseline (0.1-0.2 nmol/hr) levels. Fouriertransform infrared spectroscopy confirmed the sequestered product as Hz,as it showed characteristic absorption peaks at 1660 and 1210 cm⁻¹, aspectroscopic signature² of carboxylate side group coordinated to theiron center of ferriprotoporphyrin IX (FIG. 16 c). In vivo, Hz formationoccurs in an acidic (pH 4.5-5.2) milieu^(11.12) of the food vacuole andit was found that HDP had optimal activity in a similar environment(FIG. 16 d), that was indicative of its potential to function in thefood vacuole. It is noteworthy that HRP II (and HRP III), the only knownparasite proteins capable of Hz synthesis⁶, are only found in P.falciparum parasites, where most of the protein produced is secreted bythe parasite^(13.14) and Hz production is unaffected in parasite cloneslacking the two proteins¹⁵. This led to a suggestion that unsaturatedmembrane lipids could be producing Hz in the parasite^(7.9). However,these results clearly show that HDP is the most potent parasite factorand could be the major producer of Hz inside the parasite.

To investigate whether the heme detoxification activity demonstrated byrecombinant HDP is the true representation of its role in the parasite,native HDP from erythrocytic stage P. falciparum parasites was purified.On a SDS-PAGE gel, native HDP showed an approximate molecular weight of˜60 KDa, possibly due to dimerization, and was recognized by anti-HDPantibodies on a western blot (FIG. 16 e). Furthermore, it was found thatnative HDP was able to produce Hz at levels comparable to therecombinant protein (FIG. 16 f), which indicated that in vivo, HDP couldindeed be involved in Hz formation.

Hz formation is an indispensable step in parasite's lifecycle. Asresults from our in vitro studies inferred towards a major role for HDPin this process, its involvement was investigated in vivo by a geneticknockout experiment in erythrocytic stage P. falciparum parasites.Disruption of the HDP locus was attempted by a plasmid-based singlecross over recombination (FIG. 17 a). To promote plasmid integration atthe targeted locus, transfected parasites were subjected to three drugselection cycles over a 12 week period. In two independent experiments,parasites with a disrupted HDP locus could not be obtained and theresulting transfectants episomally carried the pHDPKO plasmid (FIG. 17b) and expressed HDP at levels comparable to the wild type parasites(not shown), Therefore, it is highly likely that HDP plays a criticalrole in Hz formation and its inactivation may not be possible.

Inside an infected erythrocyte, up to 75% of the total hemoglobin isdegraded¹⁶ giving rise to large quantities of free heme, most of whichis converted into Hz⁷. Having established the role of HDP in thisprocess, its affinity for heme was investigated by isothermal titrationcalorimetry FIG. 18 a). This interaction was studied by measuring theheat change associated with the binding of heme to HDP, at pH 5.6 whereprotein bound heme but did not make any Hz. The interaction revealed a Hof −5.03 kcal/mol, a Kd of 80 nM, and a stoichiometry (n) of 2.7 hememolecules per HDP polypeptide. This affinity is at least 4 times higherthan HRP II, whose affinity for heme is in 340-940 nM range^(18.19).

Subsequently HDP sequence were analysed for the presence of any knownheme binding motif using SMART20, a domain identification tool. WhileHDP has no homology to any of the known heme-binding proteins, theanalysis revealed that the carboxyl terminus region (amino acids 88-205)of the protein has homology (e value 3e-¹⁰) to fasciclin-l, an ancientadhesive and highly diverse domain, present in proteins of prokaryotic²¹and eukaryotic²² origin (FIG. 3). To investigate if this domain alone isresponsible for Hz formation, two truncated variants of HDP wererecombinantly produced, one encoding only the fasciclin-1 domain(residues 88-205 of SEQ ID NO: 1; protein HDP3) and the other encodingresidues 1-87 of the full length protein (i.e. of SEQ ID NO: 1, proteinHDP2) (FIG. 19 a-d). It was found that neither fasciclin-1 domain (HDP3)nor the amino terminus region (HDP2) alone were capable of Hz production(FIG. 18 b). Hence, a full length HDP is required for Hz production.

As stated earlier, HRP II and HRP III are only found in P. falciparumparasites but Hz formation occurs in all known species of Plasmodium. Toinvestigate if HDP is present in all the parasite species, the genomesof seven other species of Plasmodium ^(23.24) were examined in silico(FIG. 3). HDP orthologs were found in all the species with proteinshowing 60% sequence identity. Evidently, the protein is functionallyconserved as a recombinantly produced P. yoelii HDP generated Hz atlevels indistinguishable from its P. falciparum ortholog (FIG. 18 c).HDP seems to have an ancient lineage as its homolog was found inTheileria ²⁵ genome (FIG. 3), a hemoprotozoan that sequesters heme intonon-toxic aggregates during the intraerythrocytic stages of itslifecycle.

As Hz formation occurs inside the food vacuole, to be functionallyrelevant, one would anticipate HDP to be present inside this organelle.Though the protein lacks a classical N-terminal signal sequence or anyknown translocation signal that could predict its possible sorting andtransport to its destined site, the presence of HDP was detected insidethe food vacuole (FIG. 20 a-d). Therefore, to comprehend itsintracellular trafficking, intraerythrocytic parasites were analyzed atdifferent stages of development, for HDP expression. It was discoveredthat from the early (ring) stages of infection, HDP is secreted to thehost cell cytosol, before any detectable amount of Hz was visible insidethe parasite (FIG. 20 a). The protein accumulated inside the cytosol ofthe host cell (FIG. 20 b; FIG. 21 a-c) and was not exported out of theinfected RBC as it could not be detected in the concentrated culturesupernatant by immunoblot (data not shown). Subsequently, as parasitedevelopment progressed, it was found that HDP, along with hosthemoglobin, is trafficked to the food vacuole, through thecytostome-mediated pathway (FIG. 20 b-d; FIG. 21 a-c). By immunoelectronmicroscopy, we detected the uptake of HDP through the cytostome (FIG. 20b, FIG. 21 b), its presence in the transport vesicles (FIG. 20 c) anddelivery to the food vacuole (FIG. 20 d; FIG. 21 c). This novel andcircuitous trafficking of HDP is indicative of a functional convergencein the parasite where host hemoglobin, HDP and parasite protease26involved in hemoglobin proteolysis (and located in the vesicularmembrane), are transported together to the food vacliole.

This is the first report of a pan-Plasmodium heme detoxifying proteinthat is highly efficient in catalyzing the conversion of heme into Hz.Identification of HDP not only fills an important gap in ourunderstanding of the mechanism of Hz production in malaria parasite, butthe novel “Outbound-Inbound” trafficking of HDP also reveals aninteresting insight into the inner workings of the parasite. Due to therapid emergence of multi-drig resistant parasites, several majorantimalarial drugs have become ineffective and combination therapy isfast becoming a mainstay for malaria control²⁷. This discovery opens newavenues for designing novel antimalarial drugs that specifically targetHDP and thereby prevent the conversion of heme into Hz.

Methods for Example 9

Hz formation assay The assay was performed as previously described⁶.Briefly, equimolar amounts (0.5 nmol) of HDP, HRP II or unsaturatedlipids were added to freshly prepared heme solution in 500 mM sodiumacetate buffer pH 5.2, followed by incubation at 37° C. for 16 hrs. Thereaction was stopped by adding SDS (0.1% final conc.). Unsequesteredheme was removed by repeated washing of the pellet with 2.5% SDS and 0.1M sodium bicarbonate (pH 9.1) followed by distilled water till nosoluble heme was visible in the supernatant. Hz pellet was resuspendedin 0.1 N NaOH and absorbance was measured at 400 nm. A standard curveusing different concentrations of β-hematin was prepared to quantitatethe amount of heme incorporated into Hz. A reaction containing bufferedheme alone was used as negative control. pH dependence of HDP wasevaluated in 500 MM sodium acetate buffer of different pH (pH 3.2-6.0).All the Hz formation assays were performed at least three times intriplicates.

Purification of native HDP. Anti-HDP antibodies were raised in rabbitsand affinity purified using standard protocols. Trophozoite stage P.falciparum (3D7 strain) parasites were isolated from a 20 ml cultureusing a MACS column (Miltenyi Biotec), and resuspended in 0.2 ml ofsolubilization buffer (20 mM Tris-Cl pH 7.4, 0.5% NP-40, 1× ProteaseInhibitor Cocktail). The suspension was subjected to a singlefreeze-thaw cycle and the protein extract was clarified bycentrifugation at 15,000 g for 15 min at 4° C. Affinity purifiedanti-HDP antibodies were coupled to AminoLink® Plus Coupling Gel usingthe Seize® Primary Immunoprecipitation kit (Pierce Biotechnology), andutilized for immunoprecipitation of native HDP from the total proteinextract, as per manufacturer's instructions. Purity of the protein wasestablished by silver staining and the purified protein wasauthenticated by an ECL-based immunoblotting system (GE Health Care).

Binding affinity. Binding affinity of HDP for heme was evaluated byIsothermal titration calorimetry where freshly prepared heme solutionwas incrementally added to 5 μM HDP (in 50 mM MES, pH 5.6) presentinside the ITC cell. Data was collected at 30° C. at a 420 rpm stir rateusing 10 μl injections of the 100 μM heme into the protein solution. Theresulting measurements delta H vs. molar ratio were fit to a singlebinding site model using the MicroCal Origin analysis software.

Immunoelectron microscopy. P. falciparum infected erythrocytes werefixed in 4% paraformaldehyde/0.1% glutaraldehyde in 100 nM PIPES/0.5 mMMgCl₂, pH 7.2 for 1 hr at 4° C. and used for immunoelectron microscopyas described ²⁶. Controls omitting the primary antibody wereconsistently negative at the concentration of gold-conjugated secondaryantibodies used in these studies.

Targeted deletion of HDP P. falciparum 3D7 parasites was cultured inhuman O+erythrocytes as described previously. Ring stage parasites at10% parasitemia were transfected by electroporation with 100 μg of supercoiled pHDPKO, a pHD22Y based transfection vector containing a 509 bpfragment from the 5′ end of the HDP gene (SEQ ID NO: 2) along with humanDHFR selection cassette, using low voltage/high capacitance conditions²⁸. Transfectants were selected in the presence of 10 nM WR99210 (a giftfrom Jacobus Pharmaceuticals, Princeton N.J.) and subjected to threedrug selection cycles, each consisting of 21 days of growth in absenceof WR99210 followed by reselection of parasites in the presence of 10 nMWR99210. Genotypes were analyzed by probing blots of Eco RV-Bam HIdigested total parasite DNA, with a PCR amplified 509 bp fragment of HDPthat has been cloned in the transfection vector. The signal wasgenerated with an Alk Phos direct labelling and detection kit (GEHealthcare).

Immunofluorescence Methanol fixed smears of infected RBC at 5%parasitemia were blocked with 2.5% normal goat serum (NGS) for 30 minand incubated with rabbit anti HDP antibodies at 1:200 for 1 h. Boundantibodies were detected using fluorescein isothiocyanate(FITC)-conjugated goat anti-rabbit IgG diluted to 1:200. Parasite nucleiwere stained with 4′, 6-diamidino-2-phenylindole (DAPI). Slides weremounted with the antifade reagent (Vectorshield, KPL) and images(100×magnification) were obtained using Olympus 1×70 invertedfluorescence microscope and a Photometrix cooled charge-coupled devicecamera (CH350/LCCD) driven by DELTAVISION software from AppliedPrecision (Seattle, Wash.).

Cloning, recombinant expression and purification of HDP Coding sequenceof HDP (SEQ ID NO: 2) was amplified by RT-PCR using total RNA from theP. falciparum (3D7 strain) erythrocytic stage parasites. The amplifiedfragment was cloned in pET101, a V5 epitope and polyhistidine-tagencoding, T7 promoter-based E. coli expression vector, giving rise toplasmid pHDP. Protein, expressed in BL21 cells, was localized ininclusion bodies, which were isolated as described previously²⁹.Purified inclusion bodies were solubilized in 50 mM CAPS buffer (pH11.0) containing 1.5% N-lauryl sarkosine and 0.3 M NaCl, for 30 min andthe solubilized protein was separated by centrifugation (10,000 g; 30min). Protein was purified by affinity chromatography on His-Trap, ahigh performance nickel affinity column (GE Health Care) using animidazole gradient in 50 mM CAPS pH 11.0 containing 0.3% N-laurylsarkosine and 0.3 M NaCl. Protein-containing fractions were pooled andpurified to homogeneity by gel filtration chromatography on Superdex 20010/300 GL column (GE Health Care), equilibrated in 25 mM CAPS (pH 11.0)containing 135 mM NaCl. PyHDP (SEQ ID NO:10) was amplified by RT-PCRusing total erythrocytic stage P. yoelii RNA and cloned in pET101plasmid. Plasmids encoding protein HDP2 and HDP3 were generated bysub-cloning using pHDP as template. Their expression and purificationwas performed as described above. DNA encoding P. falciparum Histidinerich protein II was cloned in pET101 and its expression and purificationwas performed as described previously.³⁰

References for Example 9

-   1. Francis, S. E., Sullivan, D. J., Jr. & Goldberg, D. E. Hemoglobin    metabolism in the malaria parasite Plasmodium falciparum. Annu Rev    Microbiol 51, 97-123 (1997).-   2. Slater, A. F. et al. An iron-carboxylate bond links the heme    units of malaria pigment. Proc Natl Acad Sci USA 88, 325-9 (1991).-   3. Pagola, S., Stephens, P. W., Bohle, D. S., Kosar, A. D. &    Madsen, S. K. The structure of malaria pigment beta-haematin. Nature    404, 307-10 (2000).-   4. Sullivan, D. J., Jr., Gluzman, l. Y., Russell, D. G. &    Goldberg, D. E. On the molecular mechanism of chloroquine's    antimalarial action. Proc Natl Acad Sci USA 93, 11865-70 (1996).-   5. Kannan, R., Sahal, D. & Chauhan, V. S. Heme-artemisinin adducts    are crucial mediators of the ability of artemisinin to inhibit heme    polymerization. Chem Biol 9, 321-32 (2002).-   6. Sullivan, D. J., Jr., Gluzman, I. Y. & Goldberg, D. E. Plasmodium    hemozoin formation mediated by histidine-rich proteins. Science 271,    219-22 (1996).-   7. Bendrat, K., Berger, B. J. & Cerami, A. Haem polymerization in    malaria. Nature 378, 138-9 (1995).-   8. Dorn, A., Stoffel, R., Matile, H., Bubendorf, A. & Ridley, R. G.    Malarial haemozoin/beta-haematin supports haem polymerization in the    absence of protein. Nature 10374, 269-71 (1995).-   9. Fitch, C. D., Cai, G. Z., Chen, Y. F. & Shoemaker, J. D.    Involvement of lipids in ferriprotoporphyrin IX polymerization in    malaria. Biochim Biophys Acta 1454, 31-7 (1999).-   10. Gardner, M. J. et al. Genome sequence of the human malaria    parasite Plasmodium falciparum. Nature 419, 498-511 (2002).-   11. Yayon, A., Cabantchik, Z. I. & Ginsburg, H. Identification of    the acidic compartment of Plasmodium falciparum-infected human    erythrocytes as the target of the antimalarial drug chloroquine.    Embo J 3, 2695-700 (1984).-   12. Hayward, R., Saliba, K. J. & Kirk, K. The pH of the digestive    vacuole of Plasmodium falciparum is not associated with chloroquine    resistance. J Cell Sci 119, 1016-25 (2006).-   13. Akompong, T. et al. Trans expression of a Plasmodium falciparum    histidine-rich protein II (HRPII) reveals sorting of soluble    proteins in the periphery of the host erythrocyte and disrupts    transport to the malarial food vacuole. J Biol Chem 277, 28923-33    (2002).-   14. Howard, R. J. et al. Secretion of a malarial histidine-rich    protein (Pf HRP II) from Plasmodium falciparum-infected    erythrocytes. J Cell Biol 103, 1269-77 (1986).-   15. Wellems, T. E., Walker-Jonah, A. & Panton, L. J. Genetic mapping    of the chloroquine-resistance locus on Plasmodium falciparum    chromosome 7. Proc Natl Acad Sci USA 88,3382-6(1991).-   16. Goldberg, D. E., Slater, A. F., Cerami, A. & Henderson, G. B.    Hemoglobin degradation in the malaria parasite Plasmodium    falciparum: an ordered process in a unique organelle. Proc Natl Acad    Sci USA 87, 2931-5 (1990).-   17. Egan, T. J. et al. Fate of haem iron in the malaria parasite    Plasmodium falciparum. Biochem J 365, 343-7 (2002).-   18. Schneider, E. L. & Marletta, M. A. Heme binding to the    histidine-rich protein II from Plasmodium falciparum. Biochemistry    44, 979-86 (2005).-   19. Pandey, A. V. et al. Mechanism of malarial haem detoxification    inhibition by chloroquine. Biochem J 355, 333-8 (2001).-   20. Schultz, J., Milpetz, F., Bork, P. & Ponting, C. P. SMART, a    simple modular architecture research tool: identification of    signaling domains. Proc Natl Acad Sci USA 95, 5857-64 (1998).-   21. Ulstrup, J. C., Jeansson, S., Wiker, H. G. & Harboe, M.    Relationship of secretion pattern and MPB70 homology with    osteoblast-specific factor 2 to osteitis following Mycobacterium    bovis BCG vaccination. Infect Immun 63, 672-5 (1995).-   22. Kim, J. E. et al. Identification of motifs for cell adhesion    within the repeated domains of transforming growth    factor-beta-induced gene, betaig-h3. J Biol Chem 275, 30907-15    (2000).-   23. Carlton, J. M. et al. Genome sequence and comparative analysis    of the model rodent malaria parasite Plasmodium yoelii yoelii.    Nature 419, 512-9 (2002).-   24. http://www.sanger.ac.uk/Projects/Protozoa/.-   25. Pain, A. et al. Genome of the host-cell transforming parasite    Theileria annulata compared with T. parva. Science 309, 131-3    (2005).-   26. Klemba, M., Beatty, W., Gluzman, I. & Goldberg, D. E.    Trafficking of plasmepsin II to the food vacuole of the malaria    parasite Plasmodium falciparum. J Cell Biol 164, 47-56 (2004).-   27. Rathore, D., McCutchan, T. F., Sullivan, M. & Kumar, S.    Antimalarial drugs: current status and new developments. Expert Opin    Investig Drugs 14, 871-83 (2005).-   28. Fidock, D. A. & Wellems, T. E. Transformation with human    dihydrofolate reductase renders malaria parasites insensitive to    WR99210 but does not affect the intrinsic activity of proguanil.    Proc Natl Acad Sci USA 94, 10931-6 (1997).-   29. Rathore, D. et al. Molecular mechanism of host specificity in    Plasmodium falciparum infection: role of circumsporozoite protein. J    Biol Chem 278, 40905-10 (2003).-   30. Sullivan, D. J., Jr., Gluzman, I. Y. & Goldberg, D. E.    Plasmodium hemozoin formation mediated by histidine-rich proteins.    Science 271, 219-22 (1996).

Example 10 HDP as an Antimalarial Drug Target for the Two Major Species(P. falciparum & P. vivax) of Human Malaria

It has been shown that P. falciparum HDP produces hemozoin in theparasite. In this example, it is shown that the HDP ortholog from P.vivax parasites (SEQ ID NO:7) can also produce Hemozoin (FIG. 22). Theexperiment was performed as described according to methods describedabove for the previous examples. P. vivax is the second most importanthuman malaria parasite, responsible for almost 50% of the total malariacases. Though rarely lethal, it causes severe morbidity and is a majorproblem in the southeastern Asia and Latin America. Therefore, with thedemonstration that HDP from P. vivax (SEQ ID NO:7) also producesHemozoin, inhibitors of HDP developed against P. falciparum parasitecould also be used to prevent or treat P. vivax malaria infections.

Example 11 Development of HDP as a Vaccine Candidate

It has been previously shown that antibodies raised against HDP canprevent invasion of hepatocytes by P. falciparum parasites, raising thepossibility that HDP could be developed as a vaccine candidate. Thispossibility was investigated in a P. yoelii-based mouse malaria model.Both a protein and a DNA-based approach was pursued for investigatingthe potential of HDP in protecting the host from malaria. Due todifferences in haplotype, two different species of mice wereinvestigated.

Materials and Methods

Cloning of PyHDP gene from Plasmodium yoelii into a DNA vaccine plasmid,pVRJ020: RNA from Plasmodium yoelii was used for amplification of thePyHDP gene. Primers were designed to amplify a region encoding aminoacids 1 to 205 of the PyHDP gene (SEQ ID NO:9). A BamHI site [boldsequence] was incorporated into the 5′ primer(GGAATTCAGGAGCCCTTCGGATCCAAAAAAAAATTGTAT, SEQ ID NO: 40) and the 3′primer (CTTCGAATTGAGCTCGGATCCTCAAATTATTGGCTTATCTATGAT SEQ ID NO: 41).The 3′ primer also incorporated a stop codon [underlined sequence]. ThePCR fragment (618 bp) was purified using the PCR purification kit fromQiagen and digested with BamHI. The base vector pVR1020¹ containing akanamycin resistance gene was also digested with BamHI for 3 hrs at 37°C. During the last 30 min of digestion 1 unit/μl of shrimp alkalinephosphatase was added to dephosphorylate the ends of the vector. Thedigested PCR product and pVR1020 were purified after electrophoresis ona 1% agarose gel. Ligation was carried out with various vector to insertratios. The ligation was performed for 16 hrs at 14° C. Transformed Ecoli, DH5α, bacteria were plated on kanamycin selective media andincubated at 37° C. for 24 hrs. Colonies contain recombinant plasmidswere cultured and plasmids isolated. The plasmids were sequenced toconfirm the insert sequence and orientation. Plasmid clones weretransfected into VM449 cells and PyHDP expression was confirmed withwestern blot using 1:200 dilution of anti-PyHDP antibodies raised inmice. Large scale plasmid DNA from a confirmed PyHDP expressing pVRPyHDPclone was prepared and purified using an endotoxin-free plasmidpurification Giga kit (QIAGEN Inc., Valencia, Calif.).

Immunization of Mice: All animal experiments were conducted inaccordance with the guidelines indicated in the National Institutes ofHealth Guide to Laboratory Animal Care and were approved by the VirginiaTech Animal Care and Use Committee. Six week-old female BALB/c and A/jmice were used for the immunization and challenge experiment. Threegroups of eight mice each were immunized as indicated in Table 10. Thisimmunization schedule was repeated twice at intervals of 21 days for allthe groups. Groups 1 and 2 were controls for protein and plasmidimmunizations, and were immunized with PBS and base vector pVR1020,respectively. Purified recombinant PyHDP was used for subcutaneousimmunizations at 110 μg/100 μl/mouse (group 3). The first dose wasprepared in complete freunds adjuvant with subsequent doses given inincomplete freunds adjuvant. For the pVRPyHDP plasmid immunization group(group 4), DNA was injected intramuscularly (i.m.) into thegastrocnemius muscle with a 29-gauge needle using 100 μg of DNA in 100μl of phosphate-buffered saline (PBS). The last dose of the DNA vaccineimmunization regime was with recombinant PyHDP at 100 μg/100 μl/mouseprepared with incomplete freunds adjuvant and was administeredsubcutaneously.

Sporozoite preparation: Anopheles stephensi mosquitoes were reared incages at 27° C. and >80% relative humidity and were fed with 10% sucrosesolution every alternate day [2]. For the development of the sporozoitestage, mosquitoes starved of sucrose for 24 hrs, were allowed to bloodfeed on anesthetized P yoelii infected mice for 10 minutes. Samples ofsalivary glands and stomach were prepared beginning 10 days post feedingto monitor the development of the mosquito stages of the parasite.

Sporozoites were isolated using the Ozaki method ³. Briefly, on the dayof challenge (day 0) the mosquitoes were anesthetized with chloroformand thorax dissected in complete M199 medium. Crushed thorax was loadedon a silanized glass wool column prepared in Eppendorf tubes, and wascentrifuged at 2500 rpm to collect the flow through. The pellet from 2-3such tubes were resuspended and pooled. Sporozoites were counted using ahemocytometer and resuspended in complete M199 medium at a concentrationof 100 sporozoites per 100 μl. Immunized mice were challenged with 100sporozoites injected via the tail vein.

Monitoring parasitemia: Parasitemia in all mice from all the groups weremonitored on alternate days by conventional Giemsa staining ⁴ startingon day 4 after infection. Thin blood films were prepared by tailbleeding, air dried, and methanol fixed before staining.

Parasitemia was monitored for 20 days post infection or till it reached40-50%. TABLE 10 A/J BALB/c Protein Plasmid Group 1 4 4 1st: Saline +CFA [Control, 2nd: Saline + IFA Protein] 3rd: Saline + IFA Group 2 4 43rd: Saline + IFA 1st: pVR1020 [Control, 2nd: pVR1020 Plasmid] Group 3 88 1st: PyHDP + CFA [PyHDP] 2nd: PyHDP + IFA 3rd: PyHDP + IFA Group 4 8 83rd: PyHDP + IFA 1st: pVRPyHDP [pVRPyHDP + 2nd: pVRPyHDP PyHDP]Results: A/J mice immunized with DNA construct encoding PyHDP showedalmost 50% reduction of parasitemia till Day 10 post sporozoitechallenge (FIG. 23). However, animals immunized with PyHDP protein aloneshowed a marginal decrease in parasitemia (30% inhibition).Nevertheless, the initial immune response to the DNA vaccine was foundto be significant. Thus PyHDP is an ideal candidate for a subunitvaccine⁵, and in the presence of other antigens such as PFTRAP⁶ andPFCSP⁵ may generate a protective immune response. Balb/C mice immunizedwith protein or the DNA vaccine construct showed no protection⁷. andthis may be attributable to strain to strain variations in the immunesystem

References for Example 11

-   1. Price, B. M. et al. Protection against Pseudomonas aeruginosa    chronic lung infection in mice by genetic immunization against outer    membrane protein F (OprF) of P. aeruginosa. Infect Immun 69, 3510-5    (2001).-   2. Porter-Kelley, J. M. et al. Plasmodium yoelii: axenic development    of the parasite mosquito stages. Exp Parasitol 112, 99-108 (2006).-   3. Ozaki, L. S., Gwadz, R. W. & Godson, G. N. Simple centrifugation    method for rapid separation of sporozoites from mosquitoes. J    Parasitol 70, 831-3 (1984).-   4. Shute, P. G. & Maryon, M. An Improved Technique for Staining    Malaria Parasites with Giemsa Stain. Arch Roum Pathol Exp Microbiol    22, 887-94 (1963).-   5. Prieur, E. et al. A Plasmodium falciparum candidate vaccine based    on a six-antigen polyprotein encoded by recombinant poxviruses. Proc    Natl Acad Sci USA 101, 290-5 (2004).-   6. Schneider, J. et al. A prime-boost immunisation regimen using DNA    followed by recombinant modified vaccinia virus Ankara induces    strong cellular immune responses against the Plasmodium falciparum    TRAP antigen in chimpanzees. Vaccine 19, 4595-602 (2001).-   7. Belmonte, M. et al. The infectivity of Plasmodium yoelii in    different strains of mice. J Parasitol 89, 602-3 (2003).

Example 12 HTS Screening of Potential Inhibitors of P. falciparum HDP

A panel of candidate compounds were tested for their ability to inhibitHDP from P. falciparum. The protein was prepared as described above. Thetesting was carried out as follows:

HTS screening for identification of HDP inhibitors. HTS screening wasperformed at the Chemical Genomics Center of the Broad Institute ofHarvard and MIT (Cambridge, Mass.). A 2× protein stock (10 μM) wasprepared in 200 mM Sodium acetate buffer at pH 5.6. and 35 μl of thissolution was dispensed in each well of a 384 well plate, using anautomated dispenser. Through a robotized transfer mechanism involvingsteel pins, each of the protein-containing well (in a 384 well plate)received 300 nl of a compound. After the addition of the compound, theplate was incubated at room temperature for 60 minutes, followed by anaddition of 35 μl of freshly prepared heme solution at a concentrationof 20 μM. A 1:1 mix of HDP-heme gave rise to the final concentrations of5 and 10 μM of protein and heme in the reaction, respectively. Afterheme addition, the plate was incubated in dark for 60 minutes followedby the measurement of absorbance at 414 nm, utilizing a Synergy platereader integrated with a biostack. The reactions were performed induplicates and with controls, where the interactions were measured inthe absence of the protein. The readouts were stored and analyzed forthe identification of potential inhibitors of the reaction.

Data Analysis. Statistical analysis was performed utilizing acombination of parameters and compounds that showed statisticallysignificant inhibition were selected. Briefly, the backgroundabsorbance, which can be attributed to heme and compound alone andmeasured for each compound utilizing the background plate, wassubtracted from the test reads. Subsequently, the net absorbance wascompared to controls wells, that did not receive the test compounds andthe percent decrease in absorbance was measured by the followingformula:Percent inhibition=[(Absorbance in test well/Absorbance in controlwells)×100]Activity of HDP inhibitors on P. falciparum parasites. Selectedcompounds were screened for their potential to inhibit the growth of P.falciparum parasites in culture. Chloroquine sensitive 3D7 strain of theparasite was utilized for analysis. Briefly, growth of P. falciparumparasites (1% parasitemia, 1% hematocrit) in RPMI 1640 medium and 0.5%albumax was evaluated in the presence of different concentrations of theinhibitors and compared with the growth where parasites were incubatedwith medium alone. The parasites were incubated with the inhibitors for48 hours before the addition of SYBR Green dye for measuring parasitegrowth. A recently published SYBR-Green I based method was utilized forthis measurement [9]. As RBCs are terminally differentiated and lack anucleus, addition of SYBR Green to the parasite culture at the end of adesired incubation time provides a direct measurement of the DNA contentof the parasite. SYBR Green fluorescence was measured using a 384 wellplate spectrofluorometer with an excitation and emission wavelengths setat 490 and 530 nm, respectively.

Results and Conclusion

Cell-free HTS for the identification of inhibitors of HDP-Hemeinteraction. Using high throughput technology and the power ofcombinatorial chemistry, we investigated several thousand chemicalcompounds for their potential to inhibit the interactions between HDPand heme. The screening was facilitated by the knowledge that HDP, onits interaction with heme, binds to it with a very strong affinity andgives a Soret peak at 414 nm. This property of HDP was exploited fordesigning a simplified assay that could be utilized for HTS process. Atotal of 2 grams of HDP was recombinantly purified from 25 Liters of E.coli culture. The purified protein was subsequently utilized in thecell-free assay for the identification of potential inhibitors ofHDP-heme interactions. HTS was performed in 384 well plates where intypical reaction 5 μM HDP was allowed to interact with 10 μM heme in theabsence (control) or presence of excess of a chemical compound. Theconcentration of the chemical compound was in 40-50 μM range. HDP-hemeinteraction was measured at 414 nm in the presence of the compounds andcompared with control reactions that only received the carrier (DMSO).The final concentration of DMSO in the reaction was 0.4%.

A total of 110,000 drug-like, diverse heterocyclic chemical compoundswere screened during this process. These compounds were obtained fromseveral sources including established chemical vendors (Asinex,Analyticon, Biomol, Bionet, ChemDiv, Enamine, Maybridge, Spectrum,TimTec) as well as a range of diversity oriented synthesis compoundsthat have been generated by academic research laboratories from aroundthe world. Screening identified several hundred (300+) compounds (Table11) that inhibited the reaction at a statistically significant >30%levels. Successful events in this initial screen led to theconsolidation of select wells from the original library stock togenerate a new second generation of plate for screening the activity ofthese compounds on P. falciparum parasites.

Antimalarial activity of HDP-inhibitors on P. falciparum parasites. Atotal of 327 inhibitors were screened for their antimalarial activity ina P. falciparum parasite-based cellular assay. Rescreening of thesecompounds was performed at 20-40 micromolar final concentration.Parasites were incubated with the compounds for 60 hours followed by themeasurement of parasite DNA content utilizing a fluorometric assay. Theresults presented in Table 11 show the percent inhibition for compoundsat the highest concentration tested in the cell-based antimalaria assay.At the highest concentration tested, this screen identified 73 compoundsthat showed statistically significant >50% inhibition of the growth ofhuman malaria parasite in culture (Table 11).

Those of skill in the art will recognize that, while the particularcompounds in Table 11 may be utilized in the invention, versions ofthese compounds (i.e. derivatives or analogs thereof) may also bedeveloped that are optimized for in vivo use, i.e. for bioactivity. Suchoptimization may involve, for example, modifications to increase ordecrease the charge of the molecule (e.g. to increase or decreasesolubility, hydrophilicity, hydrophobicity, affinity for biologicalmembranes, etc.); to increase toxicity to the parasite; or to decreasetoxicity to the individual being treated. Such modification may alsoinvolve the substitution of charged groups (e.g. carboxyl groupsreplaced by sulfates or vice versa); the substitution or replacement ofcarbon chains (e.g. increasing or decreasing the number of carbons in analiphatic chain, introducing branched carbon chains, double bonds,triple bonds, etc. or replacing them with unbranched aliphatic chains),etc. Other modifications may include conjugation of the molecule toother entities (or to each other) to form chimeric molecules, e.g.attachment to various targeting moieties (peptides, etc.); theattachment of lipids or lipophilic moieties; conjugation to metal ions;and the like. Further, various salts of the compounds may be utilized inthe invention. All such derivatives and analogs of the compounds inTable 11 are intended to be encompassed by the present invention, solong as the resulting derivative/analog has the ability to prevent orinhibit the interaction of heme with HDP as described herein. Suchcompounds will typically be effective in at least the micromolarconcentration range, and preferably in the nanomolar concentration rangewhen administered in vivo.

Those of skill in the art will recognize that certain chemicalmodification(s) can be introduced as desired into a given compound toobtain a new derivative with modified biological properties such as:greater antimalarial potency against a particular Plasmodium sp., abroader spectrum of antimalarial activity against diverse Plasmodiumsp., enhanced oral bioavailability, less toxicity in a particular hostmammal, more advantageous pharmacokinetics and/or tissue distribution ina given host mammal, and the like. Therefore, the present inventionadditionally provides methods for obtaining such derivatives by applyingone or more well-known chemical reactions to a given compound, toprovide a derivative wherein one or more phenolic hydroxyl group(s) mayinstead be replaced by an ester, sulfonate ester, or ether group; one ormore methyl ether group(s) may instead be replaced by a phenolichydroxyl group; one or more phenolic hydroxyl group(s) may instead bereplaced be an aromatic hydrogen substituent; one or more secondaryamine site(s) may instead be replaced by an amide, sulfonamide, tertiaryamine, or alkyl quaternary ammonium salt; one or more tertiary aminesite(s) may instead by replaced by a secondary amine; and

one or more aromatic hydrogen substituent(s) may instead be replaced bya halogen, nitro, amino, hydroxyl, thiol, or cyano substituent.

Numerous references describe the process of chemoinformatics andlaboratory-based lead-optimization of pharmaceutical compounds ingeneral, or antimalarial compounds specifically, and selected referencesare incorporated herein.

-   Oprea, Tudor I. (ed.), Chemoinformatics in Drug Discovery, Methods    and Principles in Medicinal Chemistry (Volume 23), Edited by    Mannhold, Raimund/Kubinyi, Hugo/Folkers, Gerd. Wiley-VCH, Weinheim,    Germany.-   Brown, Nathan; Lewis, Richard A. Exploiting QSAR methods in lead    optimization. Current Opinion in Drug Discovery & Development    (2006), 9(4), 419-424.-   Rolf W. Winter, Jane X. Kelly, Martin J. Smilkstein, Rozalia Dodean,    Grover C. Bagby, R. Keaney Rathbun, Joshua I. Levin, David Hinrichs    and Michael K. Riscoe, Evaluation and lead optimization of    anti-malarial acridones, Experimental Parasitology, Volume 114,    Issue 1, September 2006, Pages 47-56.-   Aihua Xie, Prasanna Sivaprakasam and Robert J. Doerksen, 3D-QSAR    analysis of antimalarial farnesyltransferase inhibitors based on a    2,5-diaminobenzophenone scaffold, Bioorganic & Medicinal Chemistry,    Volume 14, Issue 21, 1 Nov. 2006, Pages 7311-7323.

References for Example 12

-   1. Francis, S. E., Sullivan, D. J., Jr. & Goldberg, D. E. (1997)    Hemoglobin metabolism in the malaria parasite Plasmodium falciparum,    Annu Rev Microbiol. 51, 97-123.-   2. Goldberg, D. E., Slater, A. F., Cerami, A. &    Henderson, G. B. (1990) Hemoglobin degradation in the malaria    parasite Plasmodium falciparum: an ordered process in a unique    organelle, Proc Natl Acad Sci USA. 87, 2931-5.-   3. Fitch, C. D., Chevli, R., Banyal, H. S., Phillips, G.,    Pfaller, M. A. & Krogstad, D. J. (1982) Lysis of Plasmodium    falciparum by ferriprotoporphyrin IX and a    chloroquine-ferriprotoporphyrin IX complex, Antimicrob Agents    Chemother. 21, 819-22.-   4. Kikuchi, G., Yoshida, T. & Noguchi, M. (2005) Heme oxygenase and    heme degradation, Biochem Biophys Res Commun. 338, 558-67.-   5. Leed, A., DuBay, K., Ursos, L. M., Sears, D., De Dios, A. C. &    Roepe, P. D. (2002) Solution structures of antimalarial drug-heme    complexes, Biochemistry. 41, 10245-55.-   6. Pandey, A. V., Bisht, H., Babbarwal, V. K., Srivastava, J.,    Pandey, K. C. & Chauhan, V. S. (2001) Mechanism of malarial haem    detoxification inhibition by chloroquine, Biochem J. 355, 333-8.-   7. Sullivan, D. J., Jr., Gluzman, I. Y., Russell, D. G. &    Goldberg, D. E. (1996) On the molecular mechanism of chloroquine's    antimalarial action, Proc Natl Acad Sci USA. 93, 11865-70.-   8. Kannan, R., Sahal, D. & Chauhan, V. S. (2002) Heme-artemisinin    adducts are crucial mediators of the ability of artemisinin to    inhibit heme polymerization, Chem Biol. 9, 321-32.

9. Bennett, T. N., Paguio, M., Gligorijevic, B., Seudieu, C., Kosar, A.D., Davidson, E. & Roepe, P. D. (2004) Novel, rapid, and inexpensivecell-based quantification of antimalarial drug efficacy, AntimicrobAgents Chemother. 48, 1807-10. TABLE 11 % Inhibition HDP- Anti- Hememalarial Plate Well SMILES Identifier IUPAC Name Interaction activity2074 F05 CN(C)c1ccc2N═c3cc(C)c(N) 42.50 100.00 cc3═Sc2c1 2075 G11COc1ccc(cc1)c2ncccc2O 2-(4-methoxyphenyl)pyridin-3-ol 61.75 95.80 2069J20 Cc1ccn2cc(nc2c1)c3ccc(O) 4-(7-methylimidazo[1,2-a]pyridin-2- 47.5093.70 c(O)c3 yl)benzene-1,2-diol 2020 P06 CCN1/C(═C/c2ccc3cc(C)2-[(Z)-(3-ethyl-6-methoxy-1,3- 58.75 93.70 ccc3[n+]2C)/Sc4cc(OC)ccc14benzothiazol-2(3H)-ylidene)methyl]- 1,6-dimethylquinolinium 2021 B19CC[n+]1c(/C═C/2\SC═CN2C) 1-ethyl-6-methoxy-4-methyl-2-[(Z)- 59.75 93.30cc(C)c3cc(OC)c4ccccc4c13 (3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium 2046 C02 Oc1ccc(cc1)c2nc(c([nH]2)4,4′-(4-phenyl-1H-imidazole-2,5- 44.00 92.90 c3ccc(O)cc3)c4ccccc4diyl)diphenol 2085 H01 Oc1cccc(Nc2nc(NCC3CCCO3)3-[(4-{[(2R)-tetrahydrofuran-2- 64.50 92.30 c4ccccc4n2)c1ylmethyl]amino}quinazolin-2- yl)amino]phenol 2105 K04 49.00 91.20 1413N13 Cc1noc(c1c2ccc3OCCCOc3c2) 4-[4-(3,4-dihydro-2H-1,5- 56.25 90.80c4ccc(O)cc4O benzodioxepin-7-yl)-3- methylisoxazol-5-yl]benzene-1,3-diol2017 B19 Clc1ccc2oc(cc(═NCc3ccco3) N-(6-chloro-2-phenyl-4H-chromen-4-45.25 90.60 c2c1)c4ccccc4 ylidene)-1-(2-furyl)methanamine 2099 E09Oc1ccc(cc1)c2sc3cc(O) [6-hydroxy-2-(4-hydroxyphenyl)-1- 49.75 90.10ccc3c2C(═O)c4ccc(OCCN5CCCCC5) benzothien-3-yl][4-(2-piperidin-1- cc4ylethoxy)phenyl]methanone 2290 H07 Oc1cc(cc(O)c1O)C(═O)1,2,3,4,6-pentakis-O-(3,4,5- 41.50 90.10 OC[C@H]2O[C@@H](OC(═O)trihydroxybenzoyl)-beta-D- c3cc(O)c(O)c(O)c3)[C@H](OC(═O) glucopyranosec4cc(O)c(O)c(O)c4) [C@@H](OC(═O)c5cc(O)c(O) c(O)c5)[C@@H]2OC(═O)c6cc(O)c(O)c(O)c6 2296 J02 O[C@H]1[C@H]2[C@H](CC(═O) 56.25 90.00O)C(═O)O[C@@H]3C(COC(═O) c4cc(O)c(O)c(O) c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O) c6cc(O)c(O)c(OC1═O)c26) [C@@H]3OC(═O)c7cc(O)c(O)c(O)c7 2078 L04 Cc1ccc2nc(cn2c1)c3ccc(O)4-(6-methylimidazo[1,2-a]pyridin-2- 46.50 89.70 c(O)c3yl)benzene-1,2-diol 2011 B03 CC(C)Nc1ccc(Nc2ccnc3cc4ccccc4cc23)N-benzo[g]quinolin-4-yl-N′- 59.00 89.00 cc1 isopropylbenzene-1,4-diamine2168 C04 OCCOc1ccc(CN2CCC[C@H](C2) 2-{(3R)-1-[4-(2- 37.75 89.00N3C(═O)c4ccccc4C3═O) hydroxyethoxy)benzyl]piperidin-3-yl}- cc11H-isoindole-1,3(2H)-dione 2080 F20 Clc1ccc(CCN2COc3ccc(Cl)6-chloro-3-[2-(4-chlorophenyl)ethyl]- 57.75 88.90 cc3C2)cc13,4-dihydro-2H-1,3-benzoxazine 1446 P02 Cc1cc(Nc2cc(Cl)cc(Cl)c2)N4-(3,5-dichlorophenyl)-6- 48.00 88.90 nc(N)n1methylpyrimidine-2,4-diamine 2019 C14 CCOC(═O)c1c(c2ccccc2) (ethyl1-benzyl-4- 49.25 88.70 n(Cc3ccccc3)c4ccc(O)c(CN(C)[(dimethylamino)methyl]-5-hydroxy- C)c142-phenyl-1H-indole-3-carboxylate 2144 I02 48.25 88.70 2016 E14COc1ccc(cc1)c2c/c(═NCCc3ccccc3)/ N-[(4E)-2-(4-methoxyphenyl)-6- 45.0088.50 c4cc(C)ccc4o2 methyl-4H-chromen-4-ylidene]-2- phenylethanamine1406 N16 Cc1nc2ccccn2c1C3(O)C(═O) (3R)-5,7-dichloro-3-hydroxy-3-(2-65.00 88.50 Nc4c3cc(Cl)cc4Cl methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one 1399 E08 OC(Cn1c(═N)sc2ccccc12)(1S)-1-(3,4-dichlorophenyl)-2-(2- 50.50 88.30 c3ccc(Cl)c(Cl)c3imino-1,3-benzothiazol-3(2H)- yl)ethanol 2021 D19COc1ccc2N(C)/C(═C/c3sc4ccccc4[n+]3C)/2-[(E)-(6-methoxy-1-methylquinolin- 45.25 88.00 C═Cc2c12(1H)-ylidene)methyl]-3-methyl-1,3- benzothiazol-3-ium 1465 J19Nc1cccc(c1)C(═C2C═CC(═N) 4,4′-methylenebis(3-hydroxy-2- 40.75 88.00C═C2)c3cccc(N)c3.OC(═O) naphthoic acid)-3,3′-[(4- c1cc2ccccc2c(Cc3c(O)iminocyclohexa-2,5-dien-1- c(cc4ccccc34)C(═O)O)c1Oylidene)methylene]dianiline (1:1) 2292 G08 COc1cc(O) 44.00 87.90c-2c(CCc3cc(OC)c(OC)cc32) c1 2085 L13 CC(Nc1ncnc2ccccc12)N—[(1S)-1-phenylethyl]quinazolin-4- 38.00 87.80 c3ccccc3 amine 2020 K07Nc1oc2c(CN3CCCCCC3) 2-amino-8-(azepan-1-ylmethyl)-3- 60.75 87.60c(O)ccc2c(═O) (1,3-benzothiazol-2-yl)-7-hydroxy- c1c4nc5ccccc5s44H-chromen-4-one 1405 A21 OC(═O)c1nc2cccc3cccc([nH]1)1H-perimidine-2-carboxylic acid 54.25 87.50 c32 1422 M20CCOc1ccc(cc1)S(═O)(═O) N-(3-chloro-4-hydroxy-1-naphthyl)-4- 57.50 86.50Nc2cc(Cl)c(O)c3ccccc23 ethoxybenzenesulfonamide 2012 D08OC(Cn1c2ccccc2c3ccccc13) 1-[(2S)-3-(9H-carbazol-9-yl)-2- 87.25 86.20C[n+]4cccc5cccc(O)c45 hydroxypropyl]-8-hydroxyquinolinium 1408 C14Cc1cc(NN)nc2ccccc12 2-hydrazino-4-methylquinoline 57.75 86.00 1471 J20CN1CCN(CC1)c2ccc3N═C([NH2]c3c2) 4-[5-[5-(4-methylpiperazin-1-yl)-3H-49.00 85.80 c4ccc5N═C([NH2]c5c4)benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol- c6ccc(O)cc62-ylidene]cyclohexa-2,5-dien- 1-one 1415 J19CCOC(═O)c1c(OCC)[nH]c2c1cc(O) ethyl 2-ethoxy-5-hydroxy-1H- 50.50 85.20c3ccccc23 benzo[g]indole-3-carboxylate 1441 F14 Clc1ccccc1SCc2cccc(c2)3-(3-{[(2- 41.75 85.10 C(═O)CC#N chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile 2082 C04 Cc1ccc(O)c(CCc2ccc(O)cc2)2-[2-(4-hydroxyphenyl)ethyl]-6- 33.50 84.80 n1 methylpyridin-3-ol 2296C09 O[C@H]1[C@@H](O) 43.00 84.50 [C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O) CCc4ccc(O)cc4)c(O)c3) [C@@H]1O 1417 C16CCN1C(═O)c2cccc3c(N) 6-amino-1-ethylbenzo[cd]indol- 44.25 82.40 ccc1c232(1H)-one 2033 K22 CN(C)CCNC(═O)c1cc2CSc3cc(Cl)7-chloro-N-[2-(dimethylamino)ethyl]- 40.75 81.70 ccc3-c2s14H-thieno[3,2-c]thiochromene-2- carboxamide 599 O10O═C1/C(═C\Nc2cccnc2)/ (2E)-2-[(pyridin-3- −123.00 80.30 Sc3ccccc13ylamino)methylene]-1- benzothiophen-3(2H)-one 2041 M08FC(F)(F)c1cccc(NC(═O) 3-[4-(9H-fluoren-9-yl)piperazin-1-yl]- 48.75 79.60CCN2CCN(CC2)C3c4ccccc4-c5ccccc35) N-[3- c1(trifluoromethyl)phenyl]propanamide 1464 H07Oc1ccc(cc1n2c(═O)[nH]c3cc(ccc23) 1-[2-hydroxy-5- 55.50 79.40C(F)(F)F)C(F)(F)F (trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H- benzimidazol-2-one 1439 119Oc1ccccc1C(═O)NC(═O) N-(3-furylcarbonyl)-2- 51.00 77.60 c2ccoc2hydroxybenzamide 2073 I04 Nc1cc(c2ccccc2)c3ccccc3n14-phenylquinolin-2-amine 38.25 77.40 2105 E08 41.75 75.80 1438 F17Oc1ccccc1C(═O)NC(═O) N-(2-hydroxybenzoyl)-2- 60.75 75.40 c2cccs2thiophenecarboxamide 1442 N18 Clc1ccc2c(Nc3ccccc3)7-chloro-N-phenylquinolin-4-amine 53.25 74.30 ccnc2c1 2160 E04Oc1cc(O)c2c(═O)cc(oc2c1) 5,7-dihydroxy-2-(3,4,5- 69.20 74.00c3cc(O)c(O)c(O)c3 trihydroxyphenyl)-4H-chromen-4- one 1447 D13COc1ccc(O)c(c1)C(═O) 3-(2-hydroxy-5-methoxybenzoyl)-2- 81.00 73.60C2N(C(═O)c3ccccc23)c4ccc(C) (4-methylphenyl)isoindolin-1-one cc4 1439G09 CCCc1ccc(cc1)C(═O)NC(═O) 2-hydroxy-N-(4- 43.25 69.80 c2ccccc2Opropylbenzoyl)benzamide 1464 J17 OC(COc1cccc2[nH]c(C#N) 4-({(2S)-3-[4-61.50 68.70 cc12)CN3CCN(CC3)C(c4ccccc4)(diphenylmethyl)piperazin-1-yl]-2- c5ccccc5hydroxypropyl}oxy)-1H-indole-2- carbonitrile 1469 D18Oc1ccc2c(═O)c(O)c(oc2c1) 2-(3,4-dihydroxyphenyl)-3,7- 46.75 68.50c3ccc(O)c(O)c3 dihydroxy-4H-chromen-4-one 2144 K02 44.50 68.30 596 G10Oc1cc(Cl)c([N+](═O)[O—]) (3aR,4R,9bR)-8-chloro-4-(4- 43.48 67.40c2C3C═CCC3C(Nc12) chlorophenyl)-9-nitro-3a,4,5,9b- c4ccc(Cl)cc4tetrahydro-3H-cyclopenta[c]quinolin- 6-ol 1409 B15Oc1ccc(Cl)cc1C(═O)c2cc(C(═O) 5-(5-chloro-2-hydroxybenzoyl)-2- 42.0066.50 Nc3ccccc3)c(═O)n(c2) oxo-N,1-diphenyl-1,2- c4ccccc4dihydropyridine-3-carboxamide 599 O09 Oc1c(Cl)cc(Br)(2E,5E)-5-(5-bromo-3-chloro-2- 43.18 66.30cc1/C═C\2/S/C(═N/c3ccccc3)/NC2═O hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one 2131 A18 Oc1ccccc1NC(═O)CCCCCCC(═O)8-[(2E)-2-(2- 42.00 66.10 N/N═C/c2ccccc2Brbromobenzylidene)hydrazino]-N-(2- hydroxyphenyl)-8-oxooctanamide 1410N17 CC(C)(c1ccc(O)c(Cl)c1) 4,4′-propane-2,2-diylbis(2- 46.00 65.30c2ccc(O)c(Cl)c2 chlorophenol) 1438 J17 COc1cccc(c1)C(═O)NC(═O)N-(2-hydroxybenzoyl)-3- 74.75 64.60 c2ccccc2O methoxybenzamide 587 L11Oc1ccc(cc1)C2═NN(C(C2) 4-[(5S)-5-(4-fluorophenyl)-1-(4- −136.50 64.50c3ccc(F)cc3)c4ccc(cc4)[N+](═O) nitrophenyl)-4,5-dihydro-1H-pyrazol- [O—]3-yl]phenol 1409 J07 Cc1ccc(cc1)n2cc(cc(C#N)5-(2-hydroxy-5-methylbenzoyl)-1-(4- 72.75 63.80 c2═O)C(═O)c3cc(C)ccc3Omethylphenyl)-2-oxo-1,2- dihydropyridine-3-carbonitrile 2086 H09Oc1c(CN2CCOCC2) 2-(morpholin-4-ylmethyl)-1-naphthol 80.00 62.90ccc3ccccc13 1424 H16 CN(C)c1ccc(cc1) 4-(1H-benzimidazol-2-yl)-N,N- 43.7562.10 c2nc3ccccc3[nH]2 dimethylaniline 1439 I09 CCc1ccc(cc1)C(═O)NC(═O)N-(4-ethylbenzoyl)-2- 51.25 60.10 c2ccccc2O hydroxybenzamide 2004 D17COc1ccc(cc1)c2nc(═O) 2-(4-methoxyphenyl)-4H-1,3- 51.25 59.50 c3ccccc3o2benzoxazin-4-one 1413 P10 CCCc1cc2c(═O)c(c(C)oc2cc1O)7-hydroxy-2-methyl-6-propyl-3- 37.75 54.70 c3ccccn3pyridin-2-yl-4H-chromen-4-one 2133 A16 Oc1ccccc1NC(═O)CCCCCC(═O)7-[(2E)-2-(biphenyl-4- 49.50 54.30 N/N═C/c2ccc(cc2)ylmethylene)hydrazino]-N-(2- c3ccccc3 hydroxyphenyl)-7-oxoheptanamide2144 E02 44.25 54.20 2047 H22 CCc1ccccc1NCc2c(O)c(C)4-{[(2-ethylphenyl)amino]methyl}-5- 44.00 53.00 ncc2CO(hydroxymethyl)-2-methylpyridin-3-ol 1408 D13 CCOc1ccccc1NC(═O)N-(2-ethoxyphenyl)-2- 75.50 52.80 c2ccccc2O hydroxybenzamide 1463 A19Oc1ccccc1C#CC#Cc2ccccc2O 2,2′-buta-1,3-diyne-1,4-diyldiphenol −82.2551.90 2035 C06 CC1CN(C(═O)Nc2ccc(Cl) (2S)-N-(4-chlorophenyl)-2-methyl-37.00 51.20 cc2)c3ccccc3O1 2,3-dihydro-4H-1,4-benzoxazine-4- carboxamide1397 L19 COc1ccc(c2[nH]nc(c2c3cc4ccccc4o3) 2-[4-(1-benzofuran-2-yl)-3-53.75 50.70 C(F)(F)F)c(O)c1 (trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol 2131 D05 Oc1ccccc1NC(═O)CCCCCCC(═O)N-(2-hydroxyphenyl)-8-[(2E)-2-(1- 51.25 49.80 N/N═C/c2cccc3ccccc23naphthylmethylene)hydrazino]-8- oxooctanamide 1442 J20Clc1ccc2c(Nc3ccc4OCOc4c3) N-(1,3-benzodioxol-5-yl)-7- 59.00 49.50ccnc2c1 chloroquinolin-4-amine 2015 G08 COc1ccc(/N═c/2cc(oc3ccc(O)(4E)-2-(4-methoxyphenyl)-4-[(4- 55.25 49.30 cc23)c4ccc(OC)cc4)cc1methoxyphenyl)imino]-4H-chromen- 6-ol 2131 C18Oc1ccccc1NC(═O)CCCCCCC(═O) 8-{(2E)-2-[(6-bromo-1,3- 66.25 49.20N/N═C/c2cc3OCOc3cc2Br benzodioxol-5- yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-8-oxooctanamide 2131 C14 COc1ccc(Br)cc1/C═N/NC(═O)8-[(2E)-2-(5-bromo-2- 77.25 48.30 CCCCCCC(═O)methoxybenzylidene)hydrazino]-N- Nc2ccccc2O(2-hydroxyphenyl)-8-oxooctanamide 591 D04Oc1ccc(/C═C/2\S\C(═N\c3ccccc3Cl)\ (2E,5Z)-2-[(2-chlorophenyl)imino]-5-42.88 48.20 NC2═O)cc1[N+](═O) (4-hydroxy-3-nitrobenzylidene)-1,3- [O—]thiazolidin-4-one 2008 J17 CCN(CC)c1ncnc2c3cc(C)N,N-diethyl-8-methyl-5H- 37.25 47.20 ccc3[nH]c12pyrimido[5,4-b]indol-4-amine 2086 M07 OC(═O)CC1Nc2ccccc2NC1═O[(2R)-3-oxo-1,2,3,4- 73.00 47.10 tetrahydroquinoxalin-2-yl]acetic acid1453 B20 46.50 46.20 2133 N23 Oc1ccccc1NC(═O)CCCCCC(═O)7-{(2E)-2-[(2-fluorobiphenyl-4- 74.00 45.80 N/N═C/c2ccc(c(F)c2)yl)methylene]hydrazino}-N-(2- c3ccccc3 hydroxyphenyl)-7-oxoheptanamide2027 M05 CCC(C)NC(═O)c1cc2cc3cc(OC) 6-methoxy-N-[(1S)-1- 42.75 44.90ccc3nc2o1 methylpropyl]furo[2,3-b]quinoline-2- carboxamide 2003 B09OC(CNCc1ccccc1)Cn2c3ccc(Cl) (2R)-1-(benzylamino)-3-(3,6- 60.25 44.60cc3c4cc(Cl)ccc24 dichloro-9H-carbazol-9-yl)propan-2- ol 1439 G19Cc1nc(sc1C(═O)NC(═O)c2ccccc2O) 2-hydroxy-N-[(4-methyl-2-phenyl- 71.2544.20 c3ccccc3 1,3-thiazol-5-yl)carbonyl]benzamide 1453 F18 46.25 43.30588 K12 OC(═O)CN1C(═O)/C(═C\c2cc(Br) [(5E)-5-(5-bromo-3-chloro-2- 49.3943.10 cc(Cl)c2O)/SC1═S hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]acetic acid 2009 A02 Fc1cccc(c1)N2C(═O)NC(═O)/(3-{(E)-[1-(3-fluorophenyl)-2,4,6- −130.50 42.90C(═C\c3cn(CC#N)c4ccccc34)/ trioxotetrahydropyrimidin-5(2H)- C2═Oylidene]methyl}-1H-indol-1- yl)acetonitrile 1364 C17Oc1cc(O)c2C(CC(═O)Oc2c1) (4S)-5,7-dihydroxy-4- 41.00 42.80 c3ccccc3phenylchroman-2-one 1408 P11 Cc1ccc(C)c(NC(═O)c2ccccc2)N-(2,5-dimethylphenyl)benzamide 55.00 41.80 c1 1394 O14NNc1nc(cc(n1)c2ccccc2) 2-hydrazino-4,6-diphenylpyrimidine 59.25 41.30c3ccccc3 2100 K09 CN(C)CCCC[C@@H]1NC(═O) allyl(3R,3′R,4′S,6′R,8′S,8a′S)-6′-{4- 44.00 40.90[C@@H]2C[C@@H]([C@@H](N2C1═O) [2-({[(3S,6R,7S,8aS)-3-[4- c3ccc(O)(dimethylamino)butyl]-6-(4- cc3)C(═O)OCCOc4ccc(cc4) hydroxyphenyl)-1,4-[C@H]5N6[C@@H]([C@@H](C(═O) dioxooctahydropyrrolo[1,2-a]pyrazin-OCC═C)[C@]57C(═O) 7-yl]carbonyl}oxy)ethoxy]phenyl}-5-Nc8ccc(I)cc87)C(═O) iodo-1′,2-dioxo-3′,4′-diphenyl-O[C@@H]([C@@H]6c9ccccc9) 1,2,3′,4′,8′,8a′-hexahydro-1′H- c % 10ccccc %10 spiro[indole-3,7′-pyrrolo[2,1- c][1,4]oxazine]-8′-carboxylate 2069N09 Oc1ccccc1NC(═O)c2cc(NC(═O) 5-(benzoylamino)-N,N′-bis(2- 51.25 40.60c3ccccc3)cc(c2)C(═O) hydroxyphenyl)isophthalamide Nc4ccccc4O 2144 G0252.00 40.50 1412 N21 Oc1ccccc1/C═C\2/SC(═S) (5E)-3-allyl-5-(2- 60.2540.30 N(CC═C)C2═O hydroxybenzylidene)-2-thioxo-1,3- thiazolidin-4-one2057 C10 CC1(C)c2cc(Cl) 2-chloro-8-hydroxy-10,10-dimethyl- 40.25 38.40ccc2-n3c1cc(O)c(c4ccccc4)c3═O 7-phenylpyrido[1,2-a]indol-6(10H)- one1402 H20 CCc1cc2c(═O)c(cOc2cc1O) 3-(1H-benzimidazol-1-yl)-6-ethyl-7-52.00 37.40 n3cnc4ccccc34 hydroxy-4H-chromen-4-one 2001 D13Cc1ccccc1NC(═O) 3-hydroxy-N-(2-methylphenyl)-2- 51.25 37.20c2cc3ccccc3cc2O naphthamide 1364 N13 Clc1ccc(cc1)C(C#N)C(═O)(2S)-2-(4-chlorophenyl)-3-oxo-4- 59.25 36.90 Cc2ccccc2phenylbutanenitrile 1366 O19 Cc1ccc(SCC(═O)c2cc(C)c(O)1-(4-hydroxy-3,5-dimethylphenyl)-2- 47.00 35.70 c(C)c2)cc1[(4-methylphenyl)thio]ethanone 2133 M04 Oc1ccccc1NC(═O)CCCCCC(═O)7-[(2E)-2-(4-fluoro-3- 51.75 35.60 N/N═C/c2ccc(F)c(Oc3ccccc3)phenoxybenzylidene)hydrazino]-N- c2 (2-hydroxyphenyl)-7- oxoheptanamide2010 M10 Oc1ccc(/C═C/2\SC(═S) (5Z)-5-(4-hydroxybenzylidene)-3- −159.5034.40 N(CC3CCCO3)C2═O)cc1 [(2R)-tetrahydrofuran-2-ylmethyl]-2-thioxo-1,3-thiazolidin-4-one 2034 O10 Oc1ccccc1c2n[nH]c3C(═O)(4R)-5-(2-furylmethyl)-3-(2- 50.50 34.40 N(Cc4ccco4)C(c23)c5ccccc5hydroxyphenyl)-4-phenyl-4,5- dihydropyrrolo[3,4-c]pyrazol-6(1H)- one1409 J15 Oc1ccc(Br)cc1C(═O)c2cc(C#N) 5-(5-bromo-2-hydroxybenzoyl)-1-(2-74.00 33.60 c(═O)n(c2)c3ccccc3F fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile 2021 D20 CCOC(═O)c1cnc2ccc(OCC) ethyl6-ethoxy-4-{[(1R)-1- 50.25 32.70 cc2c1NC(C)CCmethylpropyl]amino}quinoline-3- carboxylate 1393 M01Cc1ccc(NC(═O)Cc2c(O) 2-(4-hydroxy-2-oxo-1,2- 36.25 32.60c3ccccc3[nH]c2═O)cc1 dihydroquinolin-3-yl)-N-(4- methylphenyl)acetamide1395 I06 Clc1ccc(cc1)c2nc(═O) 2-(4-chlorophenyl)-4H-1,3- 46.25 32.50c3ccccc3o2 benzoxazin-4-one 1414 J16 Cc1[nH]nc(c1c2nc3ccccc3s2)4-[4-(1,3-benzothiazol-2-yl)-5- 33.25 32.50 c4ccc(O)cc4Omethyl-1H-pyrazol-3-yl]benzene-1,3- diol 1398 P07CC(C)c1ccc(OCC(═O)c2ccc(O) 1-(2,4-dihydroxyphenyl)-2-(4- 37.00 32.40cc2O)cc1 isopropylphenoxy)ethanone 1407 N22 OCc1ccc(Br)cc1c2cc([nH]n2)4-bromo-2-[5-(2-furyl)-1H-pyrazol-3- 55.50 32.00 c3ccco3 yl]phenol 2100F16 C[N+](C)(C)CCCC[C@@H]1NC(═O) 4-[(3S,6S,7R,8aR)-7-{[2-(4- 45.25 31.70[C@H]2C[C@H]([C@H](N2C1═O) {(3S,3′S,4′R,6′R,8′R,8a′R)-8′- c3ccc(O)cc3)[(allyloxy)carbonyl]-5-iodo-1′,2-dioxo- C(═O)OCCOc4ccc(cc4)3′,4′-diphenyl-1,2,3′,4′,8′,8a′- [C@@H]5N6[C@H]([C@H](C(═O)hexahydro-1′H-spiro[indole-3,7′- OCC═C)[C@@]57C(═O)pyrrolo[2,1-c][1,4]oxazin]-6′- Nc8ccc(I)cc87)C(═O)yl}phenoxy)ethoxy]carbonyl}-6-(4- O[C@H]([C@H]6c9ccccc9)hydroxyphenyl)-1,4- c % 10cccc % 10 dioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trimethylbutan-1- aminium 1397 C21COc1ccc2[nH]c3c(ncnc3c2c1) 8-methoxy-N,N-dimethyl-5H- 43.75 31.60 N(C)Cpyrimido[5,4-b]indol-4-amine 2078 N15 OC(═O)CN1C(═O)/C(═C\c2ccsc2)/[(5E)-4-oxo-5-(3-thienylmethylene)- 42.25 31.60 SC1═S2-thioxo-1,3-thiazolidin-3-yl]acetic acid 1445 F04 Clc1ccc2c(ccnc2c1)7-chloro-4-piperidinoquinoline 45.50 31.40 N3CCCCC3 2015 A14COc1cc(/C═C/C(═O)\C═C\c2ccc(cc2) (1E,4E)-1-[4- −91.00 31.20 N(C)C)cc(OC)(dimethylamino)phenyl]-5-(3,4,5- c1OC trimethoxyphenyl)penta-1,4-dien-3-one 2083 D04 CCCCc1c(nc(N)c(C#N) 2-amino-5-butyl-4-(4-hydroxy-3- 40.2531.20 c1c2ccc(O)c(OC)c2)c3ccccc3 methoxyphenyl)-6- phenylnicotinonitrile2015 P11 OC(═O)c1cccc(c1) 3-{2-[(1,3-dioxo-1,3-dihydro-2H- −100.00 31.20n2cccc2C═C3C(═O)c4ccccc4C3═O inden-2-ylidene)methyl]-1H-pyrrol-1-yl}benzoic acid 1398 E06 CCOC(═O)c1ccc(NC(═O)/ ethyl4-{[(2E)-3-(2-thienyl)prop-2- 42.75 31.10 C═C/c2cccs2)cc1enoyl]amino}benzoate 1411 A03 Oc1c(Br)cc(NS(═O)(═O)N-(3-bromo-4-hydroxy-1-naphthyl)- 38.00 30.90 c2ccc(Cl)cc2)c3ccccc134-chlorobenzenesulfonamide 2290 A23 COc1cc(/C═C/2\Oc3cc(O) 74.50 30.90ccc3C2═O)ccc1O 2031 K15 COC(═O)c1ccc2O/C(═C\c3ccc(O) methyl (2Z)-2-(4-−91.00 30.90 cc3)/C(═O)c2c1 hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5-carboxylate 2009 C17 COc1ccccc1NC(═O)CC(c2ccccc2)(3R)-3-(2-hydroxy-4-methylphenyl)- 50.75 30.70 c3ccc(C)cc3ON-(2-methoxyphenyl)-3- phenylpropanamide 2069 O12 CCOC(═O)c1cnc2ccc(OCC)ethyl 4-(benzylamino)-6- 75.50 30.60 cc2c1NCc3ccccc3ethoxyquinoline-3-carboxylate 1412 F08 ClC1═C(NC(═CS1)c2ccccc2)2-chloro-5-phenyl-3-pyridin-4-yl-4H- 39.75 30.50 c3ccncc3 1,4-thiazine2003 M11 CCS(═O)(═O)c1ccc(O)c(NC(═O) N-[5-(ethylsulfonyl)-2- 49.50 30.50COc2ccc(OC)cc2)c1 hydroxyphenyl]-2-(4- methoxyphenoxy)acetamide 2297 E24OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O) 52.25 30.30cc(O)c4c3═O)c5ccc(O) cc5)[C@H](O)[C@@H](O) [C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O 1463 I17 C#Cc1ccccc1Oc2ccccc2 1-ethynyl-2-phenoxybenzene−123.75 29.80 2099 B17 CC(═CCC/C(═C/CC/C(═C/Cc1c(O)4-hydroxy-3-[(2E,6E)-3,7,11- 60.00 29.70 c2ccccc2oc1═O)/C)/trimethyldodeca-2,6,10-trien-1-yl]- C)C 2H-chromen-2-one 1410 C01Cc1ccc(cc1)S(═O)(═O) {4-[(4- 70.50 29.70 c2ccc(NN)cc2methylphenyl)sulfonyl]phenyl}hydrazine 2007 C13 CCOC(═O) ethyl 4-[(2-73.25 29.60 c1cnc2ccccc2c1NCCc3ccccc3 phenylethyl)amino]quinoline-3-carboxylate 2100 E19 CC(═O)NCCCC[C@@H]1NC(═O) (3S,6S,7R,8aR)-3-(4- 42.2529.60 [C@H]2C[C@H]([C@H](N2C1═O) acetamidobutyl)-6-(4- c3ccc(O)cc3)hydroxyphenyl)-1,4- C(═O)O dioxooctahydropyrrolo[1,2-a]pyrazine-7-carboxylic acid 2057 E17 Oc1c(Cc2ccccc2)c(═O)5-benzyl-4-hydroxy-6H-pyrido[3,2,1- 52.75 29.50 n3c4ccccc4c5cccc1c53k]carbazol-6-one 2006 P15 Oc1ccc(NS(═O)(═O)c2ccccc2)N-[3-(1,3-benzothiazol-2-ylthio)-4- 54.75 29.40 cc1Sc3nc4ccccc4s3hydroxyphenyl]benzenesulfonamide 2037 G08 COc1cc(Nc2nc(SCC(═O)1-(3,4-dihydroxyphenyl)-2-({4-[(3,5- 44.25 29.20c3ccc(O)c(O)c3)nc4ccccc24) dimethoxyphenyl)amino]quinazolin- cc(OC)c12-yl}thio)ethanone 1439 M07 Oc1ccccc1C(═O)NC(═O)N-(cyclohexylcarbonyl)-2- 39.75 28.90 C2CCCCC2 hydroxybenzamide 1364 O16On1c(nc2ccc(Cl)cc12)c3ccc(Cl) 6-chloro-2-(4-chlorophenyl)-1H- 57.5028.90 cc3 benzimidazol-1-ol 2008 G14 COc1ccc(cc1)c2oc3ncn(Cc4ccccc4)3-benzyl-5,6-bis(4- 43.50 28.80 c(═N)c3c2c5ccc(OC)methoxyphenyl)furo[2,3-d]pyrimidin- cc5 4(3H)-imine 2008 H17CN(C)c1ncnc2c3cc(C) N,N,8-trimethyl-5H-pyrimido[5,4- 61.25 28.50ccc3[nH]c12 b]indol-4-amine 2016 I20 COc1ccc(CN(C(═O)CCN2C(═O)3-(1,1-dioxido-3-oxo-1,2- 44.50 28.20 c3ccccc3S2(═O)═O)benzisothiazol-2(3H)-yl)-N-(2- c4ccccc4O)cc1 hydroxyphenyl)-N-(4-methoxybenzyl)propanamide 2049 N08 FC(F)(F)c1ccc(nc1)S(═O)(═O)2-phenyl-5-({[5- 42.50 28.00 CC2═NN(C(═O)C2) (trifluoromethyl)pyridin-2-c3ccccc3 yl]sulfonyl}methyl)-2,4-dihydro-3H- pyrazol-3-one 2043 L02Clc1ccc(SCc2cc(═O)c3c(═O) 6-{[(4-chlorophenyl)thio]methyl}-2- 45.0027.80 n([nH]c3[nH]2)c4ccccc4) phenyl-1H-pyrazolo[3,4-b]pyridine- cc13,4(2H,7H)-dione 2071 D09 Oc1ccc(Br)cc1/C═N/n2cnnc24-bromo-2-[(E)-(4H-1,2,4-triazol-4- 39.75 27.60 ylimino)methyl]phenol1463 P01 CC1(C)OCC2═C(CC(CCc3ccccc3) (5S,7R)-2,2-dimethyl-5,7-bis(2-−85.25 27.40 OC2CCc4ccccc4)O1 phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3-d][1,3]dioxine 1397 C13 Cc1cc(O)n(n1)c2cccc(c2)C(F)3-methyl-1-[3- 54.75 27.00 (F)F (trifluoromethyl)phenyl]-1H-pyrazol-5-ol 2027 M11 N═C/1N2N═CSC2═NC(═O)\ (6E)-5-imino-6-{[1-(2-naphthyl)-1H-−109.50 26.90 C1═C\c3cccn3c4ccc5ccccc5c4pyrrol-2-yl]methylene}-5,6-dihydro-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidin- 7-one 2294 A05OC1[C@H](OC(═O)c2cc(O) 50.75 26.70 c(O)c(O)c2)OC3COC(═O) c4cc(O)c(O)c(O)c4-c5c(O)c(O)c(O)cc5C(═O) O[@@H]1[C@@H]3OC(═O) c6cc(O)c(O)c(O)c6c7c(O)c(O)c(O)cc7C(═O)O 1465 K07 CCC(C)[C@@H](CO[C@@H](Cc1ccccc1) isopropyl(2S)-2-{[(2S)-2-{[(2S,3R)- 37.00 26.70 C(═O) 2-{[(2S)-2-amino-3-N[C@@H](CCS(═O)(═O)C)C(═O) mercaptopropyl]amino}-3- OC(C)C)NC[C@@H](N)CSmethylpentyl]oxy}-3- phenylpropanoyl]amino}-4- (methylsulfonyl)butanoate1410 K11 COc1ccc(/C═C/2\C(═O)N(C) (3Z)-3-(3-hydroxy-4- 52.75 26.70c3ccccc23)cc1O methoxybenzylidene)-1-methyl-1,3- dihydro-2H-indol-2-one2004 P09 CCOC(═O)c1c(oc2ccc(O) ethyl 4-{[(2-anilino-2- 49.75 26.60c(CSCC(═O)Nc3ccccc3)c12) oxoethyl)thio]methyl}-5-hydroxy-2- c4ccccc4phenyl-1-benzofuran-3-carboxylate 2296 G07 O[C@H]1[C@@H](O) 45.75 25.90[C@@H](COC(═O)c2cc(O)c(O) c(O)c2)O[C@@H](Oc3ccc(C(═O)/C═C/c4ccccc4)c(O)c3) [C@@H]1O 2084 H14 CCOc1ccc2[nH]c(═O)3-benzyl-6-ethoxy-4- 71.00 25.90 c(Cc3ccccc3)c(O)c2c1hydroxyquinolin-2(1H)-one 1442 L20 Cc1ccc(Nc2ccnc3cc(Cl)ccc23)7-chloro-N-(3-fluoro-4- 70.25 25.90 cc1F methylphenyl)quinolin-4-amine1465 N16 NS(═O)(═O)c1cc2c(N═CNS2(═O) 6-chloro-2H-1,2,4-benzothiadiazine-38.50 25.80 ═O)cc1Cl 7-sulfonamide 1,1-dioxide 2075 O10CN(C)c1ccc(cc1)C(═NO) bis[4- −119.75 25.70 c2ccc(cc2)N(C)C(dimethylamino)phenyl]methanone oxime 2037 B18 CCC(Cc1ccccc1)NC(═O)N-[(1S)-1-benzylpropyl]-6-[(4- 38.25 25.50 c2c[nH]c3ccc(cc3c2═O)S(═O)methylpiperidin-1-yl)sulfonyl]-4-oxo- (═O)N4CCC(C)CC41,4-dihydroquinoline-3-carboxamide 2042 N03 COc1ccc(CCN2COc3c(C)3-[2-(4-methoxyphenyl)ethyl]-10- 41.50 25.50c4oc(═O)cc(c5ccccc5)c4cc3C2) methyl-6-phenyl-3,4-dihydro-2H,8H- cc1chromeno[6,7-e][1,3]oxazin-8-one 597 H21 Oc1ccccc1C2CC(═NN2c3ccc(cc3)2-[(5S)-1-(4-nitrophenyl)-3-phenyl- −158.75 25.30 [N+](═O)[O—])4,5-dihydro-1H-pyrazol-5-yl]phenol c4ccccc4 2015 P13Cc1cc(l)ccc1n2nc(cc2O)C(F) 1-(4-iodo-2-methylphenyl)-3- 41.00 25.30 (F)F(trifluoromethyl)-1H-pyrazol-5-ol 1398 K17 COC(═O)c1c(C)cc(O) methyl1-hydroxy-3- 41.25 25.20 n2c3ccccc3nc12methylpyrido[1,2-a]benzimidazole-4- carboxylate 2098 D22 OCCOc1ccc(cc1)(3R,3′R,4′S,6′R,8′S,8a′S)-5-(4- −84.25 25.10 [C@H]2N3[C@@H]([C@@H](C(═O)hydroxybut-1-yn-1-yl)-6′-[4-(2- O)[C@]24C(═O)Nc5ccc(C#CCCO)hydroxyethoxy)phenyl]-1′,2-dioxo- cc54)C(═O)3′,4′-diphenyl-1,2,3′,4′,8′,8a′- O[C@@H]([C@@H]3c6ccccc6)hexahydro-1′H-spiro[indole-3,7′- c7ccccc7pyrrolo[2,1-c][1,4]oxazine]-8′- carboxylic acid 2290 N23COc1cc(ccc1O)c2oc3cc(O) 42.50 25.10 cc(O)c3c(═O)c2O 2094 L02CCc1cccc(NC(═O) methyl 2,3-bis-O-(biphenyl-2- −133.75 24.90O[C@@H]2[C@@H](CO)O[C@H](OC) ylcarbamoyl)-4-O-[(3- [C@@H](OC(═O)ethylphenyl)carbamoyl]-alpha-L- Nc3ccccc3c4ccccc4)[C@H]2OC(═O)idopyranoside Nc5ccccc5c6ccccc6)c1 2079 D18 COc1ccccc1c2nnc(o2)2-[5-(2-methoxyphenyl)-1,3,4- 50.25 24.20 c3ccccc3Ooxadiazol-2-yl]phenol 2043 M16 CC(Nc1nc2ccccc2n1CC═C)2-{(1R)-1-[(1-allyl-1H-benzimidazol- 37.50 24.10 c3cc(Cl)ccc3O2-yl)amino]ethyl}-4-chlorophenol 2086 O22 CC(C)C1NC(Cc2c1[nH]c3ccccc23)(1R,3R)-1-isopropyl-2,3,4,9- 71.00 23.90 C(═O)Otetrahydro-1H-beta-carboline-3- carboxylic acid 1434 C13Cn1ncc(c1N)c2nc(cs2) 4-[4-(4-chlorophenyl)-1,3-thiazol-2- 44.50 23.70c3ccc(Cl)cc3 yl]-1-methyl-1H-pyrazol-5-amine 2018 A06OC(═O)c1ccc(N/C═C/C(═O) 4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en- 63.50 23.20c2ccco2)cc1 1-yl]amino}benzoic acid 2040 N04 Oc1c(CC(═O)NCc2ccccc2Cl)N-(2-chlorobenzyl)-2-(4-hydroxy-2- 39.50 23.10 c(═O)[nH]c3ccccc13oxo-1,2-dihydroquinolin-3- yl)acetamide 1441 N12FC(F)(F)c1cccc(SCc2cccc(c2) 3-oxo-3-[3-({[3- 48.00 23.00 C(═O)CC#N)c1(trifluoromethyl)phenyl]thio}methyl)phenyl] propanenitrile 2057 E08CCc1c(O)c(Cc2ccccc2)c(═O) 3-benzyl-5-ethyl-4-hydroxy-6-phenyl- 46.5022.80 n(c3nccs3)c1c4ccccc4 1-(1,3-thiazol-2-yl)pyridin-2(1H)-one 2290K17 Oc1ccc2C(═O)/C(═C/c3ccc(O) 49.00 22.80 c(O)c3)/Oc2c1 2026 E05OC1C(NN═C1c2ccccc2) (4S,5S)-3,5-diphenyl-4,5-dihydro- 49.25 22.70c3ccccc3 1H-pyrazol-4-ol 1407 H22 Cc1ccc(c2cc([nH]n2)c3cccs3)5-methyl-2-[5-(2-thienyl)-1H-pyrazol- 47.25 22.70 c(O)c1 3-yl]phenol2079 J15 Sc1nc(SCCOc2ccccc2) 2-[(2-phenoxyethyl)thio]quinazoline- 60.5022.70 nc3ccccc13 4-thiol 1409 C22 Cc1ccc2[nH]c(═NC(═O)N-(6-methyl-1,3-benzothiazol-2(3H)- 40.00 22.60 c3cccs3)sc2c1ylidene)thiophene-2-carboxamide 588 K09 Oc1c(/C═C/2\S\C(═N/c3ccccc3Cl)\(2Z,5Z)-2-[(2-chlorophenyl)imino]-5- 45.30 22.60 NC2═O)cccc1[N+](═O)(2-hydroxy-3-nitrobenzylidene)-1,3- [O—] thiazolidin-4-one 2005 F12COc1cc(/C═C/2\SC(═S) (2R)-2-[(5Z)-5-(4-hydroxy-3,5- 52.00 22.20N(C(C(C)C)C(═O)O)C2═O)cc(OC) dimethoxybenzylidene)-4-oxo-2- c1Othioxo-1,3-thiazolidin-3-yl]-3- methylbutanoic acid 2027 G14Oc1cc(nn1c2ccccc2) N-(3-chlorophenyl)-4-(5-hydroxy-1- 49.00 22.00C3CCN(CC3)C(═S)Nc4ccccc(Cl)c4 phenyl-1H-pyrazol-3-yl)piperidine-1-carbothioamide 1396 O08 CCc1cc(C(═O)Cc2nc3ccccc3n2C)1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1- 46.50 22.00 c(O)cc1Omethyl-1H-benzimidazol-2- yl)ethanone 1405 B15 CN(C)S(═O)(═O)c1ccc(cc1)N,N-dimethyl-4-(6- 40.50 21.60 c2cn3cc(C)ccc3n2methylimidazol[1,2-a]pyridin-2- yl)benzenesulfonamide 2049 D18CN(C)/C═C/1\N═C(OC1═O) (4Z)-2-[2-(4-chlorophenoxy)pyridin- 66.75 21.50c2cccnc2Oc3ccc(Cl)cc3 3-yl]-4-[(dimethylamino)methylene]-1,3-oxazol-5(4H)-one 1442 I17 Oc1cc(nc2ccc(Br)cc12)C(F)6-bromo-2-(trifluoromethyl)quinolin- 43.00 21.40 (F)F 4-ol 1409 L21CC1SC(═S)NN1c2ccccc2 (5R)-5-methyl-4-phenyl-1,3,4- 114.25 21.30thiadiazolidine-2-thione 1404 P06 Oc1cccnc1NC(═O)N-(3-hydroxypyridin-2-yl)-4- 70.00 21.30 c2ccc(Oc3ccccc3)cc2phenoxybenzamide 2072 D14 Cc1ccc(CCSc2ccc(Cl)c(Cl)5-{2-[(3,4-dichlorophenyl)thio]ethyl}- 69.75 21.20 c2)cn12-methylpyridine 1449 E20 COC(═O)c1cc(O)n(n1) methyl 5-hydroxy-1-[4-44.50 21.20 c2ccc(cc2)C(F)(F)F (trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate 2060 M02 CC/1Sc2ccc(Cl)cc2C(═O)\ (2R,3Z)-6-chloro-3- 43.0021.20 C1═C\N(C)C [(dimethylamino)methylene]-2-methyl-2,3-dihydro-4H-thiochromen- 4-one 1442 N22 CCC(═O)N1CCN(CC1)1-[4-(7-chloroquinolin-4- 41.75 21.20 c2cccnc3cc(Cl)ccc23yl)piperazino]propan-1-one 1410 G09 CC1(C)CC(═O)C(CC(═O) N-[2-chloro-5-43.25 20.90 Nc2cc(ccc2Cl)C(F)(F)F)C(═O) (trifluoromethyl)phenyl]-2-(4,4-C1 dimethyl-2,6- dioxocyclohexyl)acetamide 2058 D04Oc1c(c2ccccc2)c(═O)[nH]c3ccc(F) 6-fluoro-4-hydroy-3-phenylquinolin-43.75 20.60 cc13 2(1H)-one 2074 H22 Cc1cc(═O)[nH]c(SCC(═O)N-(4-bromophenyl)-2-[(3-cyano-4- 59.00 20.40 Nc2ccc(Br)cc2)c1C#Nmethyl-6-oxo-1,6-dihydropyridin-2- yl)thio]aectamide 2027 G19CCc1ccccc1NS(═O)(═O) 6-{[(2-ethylphenyl)amino]sulfonyl}-4- 44.50 20.00c2ccc3[nH]cc(C(═O)NCC4CCCO4) oxo-N-[(2S)-tetrahydrofuran-2- c(50 O)c3c2ylmethyl]-1,4-dihydroquinoline-3- carboxamide 2022 D10CC(C)NC(═O)C1(O)N(C(═O) (4R)-3-(3,4-dichlorophenyl)-4- 49.00 19.80Nc2ccccc21)c3ccc(Cl)c(Cl) hydroxy-N-isopropyl-2-oxo-1,2,3,4- c3tetrahydroquinazoline-4- carboxamide 2049 J02 FC(F)(F)C1═NN(C(═O)C1)2-phenyl-5-(trifluoromethyl)-2,4- 41.75 19.30 c2ccccc2dihydro-3H-pyrazol-3-one 1440 M20 CCCCc1c(C)[nH]c2cc(nn2c1═O)6-butyl-2-(2-furyl)-5-methyl-4,7- 44.00 19.20 c3ccco3dihydropyrazolo[1,5-a]pyrimidin-7- one 1394 F14CCn1c2ccccc2c3cc(/C═N/n4cnnc4) N-[(1E)-(9-ethyl-9H-carbazol-3- 46.2519.00 ccc13 yl)methylene]-4H-1,2,4-triazol-4- amine 1416 K22Oc1ccc2c(cc(═O)oc2c1O) 7,8-dihydroxy-4-phenyl-2H- 51.75 19.00 c3ccccc3chromen-2-one 1413 F17 COc1ccc(c2onc(C)c2c3cscn3)5-methoxy-2-[3-methyl-4-(1,3- 39.00 18.90 c(O)c1thiazol-4-yl)isoxazol-5-yl]phenol 2104 I18 73.75 18.40 2006 I09CCN(CC)c1ccc(/C═N/CCc2ccccc2) 5-(diethylamino)-2-{(E)-[(2- 65.75 18.00c(O)c1 phenylethyl)imino]methyl}phenol 2016 H03ON(C(═O)Nc1ccccc1)c2ccc(Cl) 1-(4-chlorophenyl)-1-hydroxy-3- 67.50 17.90cc2 phenylurea 2013 K04 Cc1nn(c(O)c1Sc2ccc(Cl)cc2)4-[(4-chlorophenyl)thio]-3-methyl-1- 53.00 17.80 c3ccccc3phenyl-1H-pyrazol-5-ol 2290 N04 Oc1ccc(c(O)c1)c2oc3cc(O)2-(2,4-dihydroxyphenyl)-3,5,7- 65.00 17.80 cc(O)c3c(═O)c2Otrihydroxy-4H-chromen-4-one 2058 E18 O═C1/C(═C\c2c[nH]c3ccccc23)/(2S,3Z)-3-(1H-indol-3-ylmethylene)- −109.50 17.50 C(Oc4ccccc14)c5ccccc52-phenyl-2,3-dihydro-4H-chromen-4- one 2041 A04 CCOc1ccc(cc1)N(C)S(═O)6-{[(4- 40.75 17.30 (═O)c2ccc3[nH]cc(C(═O)ethoxyphenyl)(methyl)amino]sulfonyl}- NCC4CCCO4)c(═O)c3c24-oxo-N-[(2S)-tetrahydrofuran-2- ylmethyl]-1,4-dihydroquinoline-3-carboxamide 2010 I17 CN(C)C1OC2═C(C═C1C)C(═O)(10S)-10-(dimethylamino)-9-methyl- 48.00 17.30 c3cccc4cccc2c347H,10H-naphtho[1,8-gh]chromen-7- one 2056 P12Clc1ccc(cc1)N2N═C(CSc3nccc(n3) 2-(4-chlorophenyl)-5-{[(4-pyridin-3-61.25 17.20 c4cccnc4)CC2═O ylpyrimidin-2-yl)thio]methyl}-2,4-dihydro-3H-pyrazol-3-one 1443 J06 Oc1c(Cc2ccccc2)c(═O)3-benzyl-4-hydroxy-1,2- 63.50 16.80 [nH]c3ccccc13 dihydroquinolin-2-one2084 K01 CC1(C)CC(═O)C2═C(C1) (4S)-4-(2-furyl)-3-hydroxy-7,7- 45.0016.60 Nc3nn(c(O)c3C2c4ccco4) dimethyl-2-phenyl-2,4,6,7,8,9- c5ccccc5hexahydro-5H-pyrazolo[3,4- b]quinolin-5-one 1364 E16On1c(nc2ncccc12)c3ccc(Cl) 2-(2,4-dichlorophenyl)-1H- 61.75 16.50 cc3Climidazo[4,5-b]pyridin-1-ol 2030 M08 O═C(Nc1cccc(c1)c2cn3cccnc3n2)N-(3-imidazo[1,2-a]pyrimidin-2- 45.00 16.40 C4CCCC4ylphenyl)cyclopentanecarboxamide 2078 J10 Cc1cc(═O)oc2c(C)c(O)c(CC═C)6-allyl-7-hydroxy-4,8-dimethyl-2H- 42.25 16.20 cc12 chromen-2-one 2011L02 CCOC(═O)c1cnc2ccc(C) ethyl 6-methyl-4-[(4-morpholin-4- 44.00 16.00cc2c1Nc3ccc(cc3)N4CCOCC4 ylphenyl)amino]quinoline-3- carboxylate 2072J04 Oc1c(oc2ccccc2c1═O)c3ccc(F) 2-(4-fluoro-3-phenoxyphenyl)-3- 58.5015.90 c(Oc4ccccc4)c3 hydroxy-4H-chromen-4-one 2014 O13CCOC(═O)c1ccc(NC(═O) ethyl 4-[({[(5R)-5-ethyl-4,6-dioxo- 49.50 15.90CSC2═NC(═O)C(CC)C(═O) 1,4,5,6-tetrahydropyrimidin-2- N2)cc1yl]thio}acetyl)amino]benzoate 2069 G20 CCS(═O)(═O)c1ccc(NC(═O)N-[4-(ethylsulfonyl)-2- 43.25 15.70 c2ccccc2)c(O)c1hydroxyphenyl]benzamide 2027 E19 COc1cccc(NS(═O)(═O) N-benzyl-6-{[(3-33.75 15.50 c2ccc3[nH]cc(C(═O)N(C)Cc4ccccc4)methoxyphenyl)amino]sulfonyl}-N- c(═O)c3c2)c1methyl-4-oxo-1,4-dihydroquinoline- 3-carboxamide 2011 A15O═C(Oc1cccc(Nc2ncnc3ccccc23) 3-(quinazolin-4-ylamino)phenyl 48.75 15.40c1)c4cccs4 thiophene-2-carboxylate 2088 A12 CCOC(═O)/C═C/c1cc(ccc1O)ethyl (2E)-3-(2-hydroxy-5- 45.50 15.20 [N+](═O)[O—] nitrophenyl)acrylate1431 J17 CSCc1ccc(cc1)C(═O)Nc2ccc(C) N-(2-hydroxy-4-methylphenyl)-4-50.50 15.10 cc2O [(methylthio)methyl]benzamide 1415 K11CCc1cc(c2n[nH]cc2c3nc4ccccc4n3C) 4-ethyl-6-[4-(1-methyl-1H- 46.75 14.90c(O)cc1O benzimidazol-2-yl)-1H-pyrazol-3- yl]benzene-1,3-diol 1416 N11CCCc1cc(═O)oc2cc(O)cc(O) 5,7-dihydroxy-4-propyl-2H-chromen- 62.50 14.90c12 2-one 2017 K21 CC1═NN(C(═O)C1C(═O) (4S)-4-(2-bromobenzoyl)-5-methyl-43.25 14.60 c2ccccc2Br)c3ccccc3 2-phenyl-2,4-dihydro-3H-pyrazol-3- one1439 C09 Oc1ccccc1C(═O)NC(═O) N-[(3-chloro-1-benzothiophen-2- 73.5014.50 c2sc3ccccc3c2Cl yl)carbonyl]-2-hydroxybenzamide 1410 H21Oc1ccc(Oc2c(F)c(F)c(Oc3ccc(O) 4,4′-[(2,3,5,6-tetrafluoro-1,4- 37.5014.40 cc3)c(F)c2F)cc1 phenylene)bis(oxy)]diphenol 2012 D07COc1ccccc1CC(═O)Nc2ccc(cc2) N-[4-(1H-benzimidazol-2-yl)phenyl]- 74.0014.20 c3nc4ccccc4[nH]3 2-(2-methoxyphenyl)acetamide 2068 J16CC(C)CCCN1C(═O)NC(═O)/ (5E)-1-(4-methylpentyl)-5-(1H-pyrrol- −112.2514.00 C(═C\c2ccc[nH]2)/C1═O 2-ylmethylene)pyrimidine-2,4,6(1H,3H,5H)-trione 2293 F13 Oc1ccc(cc1)[C@H]2CC(═O) 53.25 13.90c3c(O)cc(O)c([C@H]4[C@@H](Oc5cc(O) cc(O)c5C4═O) c6ccc(O)cc6)c3O2 1393A03 Oc1c(CC(═O)Nc2ccc(F)cc2) N-(4-fluorophenyl)-2-(4-hydroxy-2- 37.7513.80 c(═O)[nH]c3ccccc13 oxo-1,2-dihydroquinolin-3- yl)acetamide 1439E21 Cc1ccc(cc1)c2nc(═O) 2-(4-methylphenyl)-4H-1,3- 52.75 13.70c3ccccc3o2 benzoxazin-4-one 1439 A09 COc1ccccc1CC(═O)NC(═O)2-hydroxy-N-[2-(2- 37.50 13.60 c2ccccc2O methoxyphenyl)acetyl]benzamide2057 M10 Oc1c(Cc2ccccc2)c(═O) 3-benzyl-4-hydroxy-1- 69.25 13.60n(c3ccccc3)c4ccccc14 phenylquinolin-2(1H)-one 2016 O15Oc1cc(c2cc(ccc2Cl)C(F)(F) 7-[2-chloro-5- 41.00 13.60 F)c3oc(═O)sc3c1(trifluoromethyl)phenyl]-5-hydroxy- 1,3-benzoxathiol-2-one 1415 K09CCc1cc2c(═O)c(c3nc4ccccc4n3C) 6-ethyl-7-hydroxy-3-(1-methyl-1H- 41.7513.50 c(oc2cc1O)C(F)(F)F benzimidazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one 2059 D01 Cc1ccc(cc1)N2C(C(═O)(3S)-3-(2-hydroxy-4-methylbenzoyl)- 51.75 13.40c3ccc(C)cc3O)c4ccccc4C2═O 2-(4-methylphenyl)isoindolin-1-one 1439 E09Cc1ccc(Oc2ncccc2C(═O) 2-hydroxy-N-{[2-(4-methylphenoxy)- 81.00 13.20NC(═O)c3ccccc3O)cc1 3-pyridinyl]carbonyl}benzamide 1413 N19Cc1csc(n1)c2c(oc3cc(O)c(C) 7-hydroxy-6-methyl-3-(4-methyl-1,3- 44.0013.00 cc3c2═O)C(F)(F)F thiazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one 2091 D09 −79.00 12.70 1465 D10 CCN(CCCc1ccccc1)N-ethyl-3-phenyl-N-(3- 43.25 12.60 CCCc2ccccc2phenylpropyl)propan-1-amine 2073 I20 CC(═O)Nc1cccc(c1O)c2cc(═O)N-[2-hydroxy-3-(4-oxo-4H-chromen- 40.50 12.60 c3ccccc3o22-yl)phenyl]acetamide 1413 L10 CCCc1cc(C(═O)Cc2ccc3OCOc3c2)2-(1,3-benzodioxol-5-yl)-1-(2,4- 55.50 12.50 c(O)cc1Odihydroxy-5-propylphenyl)ethanone 2067 O04 Cc1sc2NC(NC(═O)c2c1C)/(2R)-2-[(E)-2-(1,3-benzodioxol-5- −96.25 12.20 C═C/c3ccc4OCOc4c3yl)vinyl]-5,6-dimethyl-2,3- dihydrothieno[2,3-d]pyrimidin-4(1H)- one2081 H11 CCCc1cc(O)c2c(C)cc(═O) 5-hydroxy-4-methyl-7-propyl-2H- 63.2511.80 oc2c1 chromen-2-one 1406 M17 Oc1cc(c2ccc(Br)cc2)c3oc(═O)7-(4-bromophenyl)-5-hydroxy-1,3- 43.50 11.80 sc3c1 benzoxathiol-2-one1413 D15 Cc1cc(O)cc2oc(c(c3cnn(c3) 7-hydroxy-5-methyl-3-(1-phenyl-1H-43.00 11.70 c4ccccc4)c(═O)c12)C(F)(F)Fpyrazol-4-yl)-2-(trifluoromethyl)-4H- chromen-4-one 2014 G14CC(C)n1nc(O)c2C(SCC(═O) (4R)-4-(4-bromophenyl)-3-hydroxy- 42.25 11.70Nc21)c3ccc(Br)cc3 1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7(6H)- one 1425 M21CCNC(═O)CC1Nc2cc(C)c(C) 2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4- 48.50 11.50cc2NC1═O tetrahydroquinoxalin-2-yl]-N- ethylacetamide 2297 M15COC(═O)[C@]1(Cc2ccc(O) 49.00 11.20 c(CC═C(C)C)c2)OC(═O)C(═C1c3ccc(O)cc3)O 1412 M06 CC(C)C(═O)N-[2-(1H-benzimidazol-2-yl)phenyl]- 43.00 11.10Nc1ccccc1c2nc3ccccc3[nH]2 2-methylpropanamide 1446 D05CC1(C)c2ccccc2-n3c1cc(O) 7-benzyl-8-hydroxy-10,10-dimethyl- 53.50 11.00c(Cc4ccccc4)c3═O 6,10-dihydropyrido[1,2-a]indol-6-one 2081 P14CCCc1c(O)c2ccccc2[nH]c1═O 4-hydroxy-3-propylquinolin-2(1H)- 39.00 10.60one 1425 O02 Oc1ccc(c2n[nH]c(c2c3cnn(c3)4-[1′-phenyl-5-(trifluoromethyl)- 40.25 10.10 c4ccccc4)C(F)(F)F)c(O)c11H,1′H-4,4′-bipyrazol-3-yl]benzene- 1,3-diol 2064 B02O═C(C1═NN(C2C1C(═O) (3aS,6aS)-3-benzoyl-1,5-diphenyl- −102.50 9.80N(C2═O)c3ccccc3)c4ccccc4) 3a,6a-dihydropyrrolo[3,4-c]pyrazole- c5ccccc54,6(1H,5H)-dione 1412 J10 CSc1nc2ccc(NC(═O)c3cccc(Cl)3-chloro-N-[2-(methylthio)-1,3- 45.25 9.80 c3)cc2s1benzothiazol-6-yl]benzamide 1395 D05 CCCn1c(nc2ccccc12)c3ccc(N)4-(1-propyl-1H-benzimidazol-2- 39.25 9.60 cc3 yl)aniline 2077 D11CN(C)c1ccc(cc1)C(N2CCCCC2) 6-[(S)-[4- 46.00 9.20 c3cc4OCOc4cc3O(dimethylamino)phenyl](piperidin-1- yl)methyl]-1,3-benzodioxol-5-ol 1405H15 Cc1oc2cc(O)ccc2c(═O) 3-(4-bromophenyl)-7-hydroxy-2- 51.25 9.20c1c3ccc(Br)cc3 methyl-4H-chromen-4-one 2073 K12 CC(NC(═O)Oc1c(Cl)cc(Cl)2,4-dichloro-1-naphthyl [2,2,2- 53.00 9.20 c2ccccc12)(C(F)(F)F)C(F)(F)Ftrifluoro-1-methyl-1- (trifluoromethyl)ethyl]carbamate 2018 O08Cc1ccc(cc1)C2═C/C(═C/c3ccc(o3) 3-(5-{(Z)-[5-(4-methylphenyl)-2- 62.758.60 c4cccc(c4)C(═O)O)/ oxofuran-3(2H)-ylidene]methyl}-2- C(═O)O2furyl)benzoic acid 2010 P21 CCOC(═O)c1c(CSc2ccc(C) ethyl 6-bromo-4-51.00 8.60 cc2)n(C)c3cc(Br)c(O)c(CN(C)[(dimethylamino)methyl]-5-hydroxy- C)c13 1-methyl-2-{[(4-methylphenyl)thio]methyl}-1H-indole- 3-carboxylate 1408 L07Oc1ccccc1C(═O)Nc2cccnc2 2-hydroxy-N-pyridin-3-ylbenzamide 56.00 8.401469 I17 Oc1ccc(CCC(═O)c2c(O)cc(O) 3-(4-hydroxyphenyl)-1-(2,4,6- 45.507.90 cc2O)cc1 trihydroxyphenyl)propan-1-one 1414 B12CCS(═O)(═O)c1ccc(O)c(c1) 2-[5-(ethylsulfonyl)-2- 37.00 7.80N2C(═O)c3cccc4cccc(C2═O) hydroxyphenyl]-1H- c34benzo[de]isoquinoline-1,3(2H)-dione 1406 I10 Cc1ccc(c(C)c1)n2c(N)c(C#N)2-amino-1-(2,4-dimethylphenyl)-1H- 46.75 7.50 c3nc4ccccc4nc23pyrrolo[2,3-b]quinoxaline-3- carbonitrile 1425 A04COc1ccc(c2n[nH]c(c2c3cnn(c3) 3-methoxy-2-methyl-6-[1′-phenyl-5- 32.757.30 c4ccccc4)C(F)(F)F)c(O) (trifluoromethyl)-1H,1′H-4,4′- c1Cbipyrazol-3-yl]phenol 2018 C20 OC(═O)CC(N1C(═O)/(2S)-2-[(5E)-5-(1H-indol-3- 44.50 7.30 C(═C\c2c[nH]c3ccccc23)/SC1═S)ylmethylene)-4-oxo-2-thioxo-1,3- C(═O)O thiazolidin-3-yl]succinic acid1409 P14 Oc1ccccc1C(═O) N-benzyl-2-hydroxybenzamide 43.00 7.10NCc2ccccc2 2058 B10 Cc1nn(c(O)c1Cc2c(Cl)4-(2,6-dichlorobenzyl)-3-methyl-1- 45.50 6.90 cccc2Cl)c3ccccc3phenyl-1H-pyrazol-5-ol 2025 O12 Clc1ccccc1CNS(═O)(═O)N-(2-chlorobenzyl)-2-phenyl-1H- 45.50 6.80 c2ccc3[nH]c(nc3c2)c4ccccc4benzimidazole-5-sulfonamide 2072 F12 CCOC(═O)Oc1c(Cl)cc2oc(═O)4-bromo-6-chloro-2-oxo-1,3- 50.00 6.40 sc2c1Br benzoxathiol-5-yl ethylcarbonate 2069 I05 Cc1cccc2c(O)c(/C═C/3\C(NN(C3═O)4-hydroxy-8-methyl-3-{(E)-[(3R)-5- 47.25 6.20 c4ccccc4)c5ccccc5)oxo-1,3-diphenylpyrazolidin-4- c(═O)[nH]c12ylidene]methyl}quinolin-2(1H)-one 1441 L02 Clc1ccc(SCc2cccc(c2)C(═O)3-(3-{[(4- 42.50 6.00 CC#N)cc1 chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile 2049 B03 CN(/N═C/c1cc(Cl)cc(Cl)c1O)3,5-dichloro-2-hydroxybenzaldehyde 56.00 5.90 C(═S)NC(C)(C)CN-tert-butyl-N′- methylthiosemicarbazone 2069 I04 Oc1ccc(Cl)cc1Sc2cc(Cl)2,2′-thiobis(4-chlorophenol) 41.25 5.80 ccc2O 1414 B15CC(C)CC1C(═C(N)OC2═C1C(═O) (4S,7R)-2-amino-4-isobutyl-5-oxo-7- 62.255.40 CC(C2)c3ccccc3)C#N phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile 1409 M11 Oc1c(Cl)cc(Cl)cc1C(═O) (3,5-dichloro-2-67.50 5.40 c2cnoc2 hydroxyphenyl)(isoxazol-4- yl)methanone 1414 J04CC(C)(C)C(═O)Nc1ccc(O)c(c1) N-[3-(1,3-benzothiazol-2-yl)-4- 46.75 5.30c2nc3ccccc3s2 hydroxyphenyl]-2,2- dimethylpropanamide 1416 D17c1cnc2ccc(cc2c1) 3,6′-biquinoline 38.00 4.70 c3ccc4ncccc4c3 2011 E12CC(Oc1ccc(Cl)cc1Cl)C(═O) (2R)-2-(2,4-dichlorophenoxy)-N-(5- 62.25 4.70NC2═NN(C(═O)C2)c3ccccc3 oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)propanamide 2144 J08 45.50 3.90 1426 J16Oc1ccc(NS(═O)(═O)c2cccs2) N-[3-(1,3-benzothiazol-2-ylthio)-4- 55.75 3.90cc1Sc3nc4ccccc4s3 hydroxyphenyl]thiophene-2- sulfonamide 588 M05CCN(CC)c1ccc(/C═C\2/SC(═O) (5E)-5-[4- −138.00 3.50 N(CNc3ccccc3OC)C2═O)(diethylamino)benzylidene]-3-{[(2- cc1 methoxyphenyl)amino]methyl}-1,3-thiazolidine-2,4-dione 2058 E04 Oc1c(c2ccccc2)c(═O)4-hydroxy-5-phenyl-6H-pyrido[3,2,1- 43.75 3.10 n3c4ccccc4c5cccc1c53jk]carbazol-6-one 1443 G08 Cc1ccc(Sc2cccc3nc(N)nc(N)5-[(4-methylphenyl)thio]quinazoline- 37.50 2.50 c23)cc1 2,4-diamine 2020K14 CCc1ccc(cc1)C2C3═C(CCCC3═O) (4R)-4-(4-ethylphenyl)-3-hydroxy-2-61.00 2.30 Nc4nn(c(O)c24) phenyl-2,4,6,7,8,9-hexahydro-5H- c5ccccc5pyrazolo[3,4-b]quinolin-5-one 1424 I20 CC(═O)n1cc(c2c(O)3-(1-acetyl-1H-indol-3-yl)-4-hydroxy- 51.50 2.00 c3ccccc3oc2═O)c4ccccc142H-chromen-2-one 2081 D13 N(c1ccccc1)c2nc(nc3ccccc23)N,2-diphenylquinazolin-4-amine 50.00 1.30 c4ccccc4 1398 D22ClC1ccc(cc1)C(═O) 2-anilino-2-oxoethyl 2-(4- 58.00 0.80c2ccccc2C(═O)OCC(═O)Nc3ccccc3 chlorobenzoyl)benzoate 1394 A01Fc1ccc(cc1)C(═O)Nc2cccc(c2) 4-fluoro-N-[3- 28.00 0.00 C(F)(F)F(trifluoromethyl)phenyl]benzamide 1417 A07 *c1ccccc1C2C(═O)N(C) 34.750.00 c3ccccc3C2═O 2030 A14 C(c1ccccc1)n2cc3c(nnc3c4ccccc24)5-benzyl-3-phenyl-5H-pyrazolo[4,3- 51.00 0.00 c5ccccc5 c]quinoline 1413B06 CCCCc1cc(C(═O)Cc2ccccn2) 1-(5-butyl-2,4-dihydroxyphenyl)-2- 48.750.00 c(O)cc1O pyridin-2-ylethanone 1416 C02CCOc1ccc2C(═O)/C(═C\c3ccccc3O)/ (2E)-6-ethoxy-2-(2- 36.25 0.00 Sc2c1hydroxybenzylidene)-1- benzothiophen-3(2H)-one 1397 C08COc1ccc(/C═C/C(═O)Nc2ccc(C) (2E)-3-(3,4-dimethoxyphenyl)-N- 50.50 0.00c(C)c2)cc1OC (3,4-dimethylphenyl)acrylamide 1415 C11CCc1cc(C(═O)Cn2cnc3ccccc23) 2-(1H-benzimidazol-1-yl)-1-(5-ethyl- 42.000.00 c(O)cc1O 2,4-dihydroxyphenyl)ethanone 1422 C11COc1cc(Cn2c(nc3ccccc23) 4-[1-(4-hydroxy-3-methoxybenzyl)- 41.75 0.00c4ccc(O)c(OC)c4)ccc1O 1H-benzimidazol-2-yl]-2- methoxyphenol 1417 C14CCC(C)Sc1nnc(NC(═O) N-(5-{[(1S)-1-methylpropyl]thio}- 42.00 0.00c2ccccc2C(F)(F)F)s1 1,3,4-thiadiazol-2-yl)-2- (trifluoromethyl)benzamide1418 D05 COCC(═O)Oc1c(Sc2ccc(C) 3-methyl-4-[(4-methylphenyl)thio]-1-40.75 0.00 cc2)c(C)nn1c3ccccc3 phenyl-1H-pyrazol-5-yl methoxyacetate1469 D07 COc1cc(O)c2c(═O)c(O)c(oc2c1) 2-(3,4-dihydroxyphenyl)-3,5- 48.500.00 c3ccc(O)c(O)c3 dihydroxy-7-methoxy-4H-chromen- 4-one 1446 D07Oc1cc2nnnn2nc1c3ccccc3 6-phenyl[1,2,3,4]tetraazolo[1,5- 43.50 0.00b]pyridazin-7-ol 1412 D10 COc1ccc(cc1OC)C(═O)3,4-dimethoxy-N-(4-methyl-1,3- 53.25 0.00 Nc2nc3c(C)cccc3s2benzothiazol-2-yl)benzamide 1432 D13 Oc1c(Cc2ccccc2)c(═O)6-benzyl-7-hydroxy-2,3-dihydro- 68.75 0.00 n3CCCc4cccc1c431H,5H-pyrido[3,2,1-ij]quinolin-5-one 1408 D15 Cc1ccc(NC(═O)c2ccccc2O)2-hydroxy-N-(4- 62.25 0.00 cc1 methylphenyl)benzamide 2080 E05Cc1ccc(cc1)N2COc3ccc(Cl) 6-chloro-3-(4-methylphenyl)-3,4- 41.50 0.00cc3C2 dihydro-2H-1,3-benzoxazine 1426 F20 CCCCN1C(═O)C2ON(C(C2C1═O)(3S,3aR,6aR)-3-(5-bromo-2- 43.00 0.00 c3cc(Br)ccc3O)hydroxyphenyl)-5-butyl-2- c4ccccc4 phenyldihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione 1442 G19 Oc1cc(nc2c(OC(F)(F)F)8-(trifluoromethoxy)-2- 52.75 0.00 cccc12)C(F)(F)F(trifluoromethyl)quinolin-4-ol 1410 H10 Cc1nn(c2ccccc2)c3[nH]c(═O)3-methyl-1-phenyl-4- 40.50 0.00 cc(c13)C(F)(F)F(trifluoromethyl)-1,7-dihydro-6H- pyrazolo[3,4-b]pyridin-6-one 1438 H17Oc1ccccc1C(═O)NC(═O) N-(2-hydroxybenzoyl)-4- 58.00 0.00c2ccc(cc2)C(F)(F)F (trifluoromethyl)benzamide 1412 I04Cc1nc2ccc(NC(═O)/C═C/c3ccccc3) (2E)-N-(2-methyl-1,3-benzothiazol- 62.500.00 cc2s1 6-yl)-3-phenylacrylamide 1399 I05 NC(═O)c1ccc(NC(═O)N-(4-carbamoylphenyl)-1-phenyl-3- 39.50 0.00c2cn(nc2c3cccs3)c4ccccc4)cc1 (2-thienyl)-1H-pyrazole-4- carboxamide 1436I16 Cc1nc(nc(SCC(═O)c2ccccc2) 2-{[5-chloro-6-methyl-2-(2-pyridinyl)-45.75 0.00 c1Cl)c3ccccn3 4-pyrimidinyl]sulfanyl}-1-phenyl-1- ethanone1409 I20 Cc1ccc(cc1)S(═O)(═O) N-(5-hydroxy-1-naphthyl)-4- 44.25 0.00Nc2cccc3c(O)cccc23 methylbenzenesulfonamide 1394 I20 N/1/C(═N\c2ccccc2)/N,N′-1H-isoindole-1,3(2H)- 46.25 0.00 c3ccccc3\C1═N\c4ccccc4diylidenedianiline 1410 J05 S═c1cc(sc2ccccc12)2-phenyl-4H-thiochromene-4-thione 45.50 0.00 c3ccccc3 1416 J09CCC1CCCCN1Cc2c(O)cc(C) 4-{[(2S)-2-ethylpiperidin-1-yl]methyl}- 39.000.00 c3c4ccccc4c(═O)oc23 3-hydroxy-1-methyl-6H- benzo[c]chromen-6-one1396 J14 CCCn1c(/N═C/c2c[nH]c3ccccc23)N-[(1E)-1H-indol-3-ylmethylene]-1- 66.00 0.00 nc4ccccc14propyl-1H-benzimidazol-2-amine 1439 K19 Oc1ccccc1C(═O)NC(═O)N-(2,3-dihydro-1-benzofuran-5- 80.00 0.00 c2ccc3OCCc3c2ylcarbonyl)-2-hydroxybenzamide 1408 L13 Oc1ccc(Cl)cc1C(═O)5-chloro-2-hydroxy-N- 42.25 0.00 Nc2ccccc2 phenylbenzamide 1415 L17COc1cccc(c1)C(═O) 3-methoxy-N-(3-[1,3]oxazolo[4,5- 36.00 0.00Nc2cccc(c2)c3nc4ncccc4o3 b]pyridin-2-ylphenyl)benzamide 2293 L18O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O) 47.25 0.00 cc(O)c4C3═O)c5ccc(O)cc5) c(O)cc(O)c2C1═O)c6ccc(O) cc6 1415 L21CC(═O)N/C(═C\c1ccccc1)/ (2Z)-2-acetamido-N-(3,5- 48.00 0.00C(═O)Nc2cc(C)cc(C)c2 dimethylphenyl)-3-phenylacrylamide 1404 M10Cc1cc(═O)[nH]c(SCC(═O) 2-[(3-cyano-4-methyl-6-oxo-1,6- 60.25 0.00Nc2cccc3ccccc23)c1C#N dihydropyridin-2-yl)thio]-N-1- naphthylacetamide593 O09 COc1cccc(/C═C\2/S/C(═N\c3cc(C) (2Z,5E)-2-[(3,5- −1.00 0.00cc(C)c3)/NC2═O)c1O dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)-1,3- thiazolidin-4-one 2073 O11Cn1c(═O)n(C)c2c(O)c([nH]c2c1═O) 6-(4-chlorophenyl)-7-hydroxy-1,3- 42.500.00 c3ccc(Cl)cc3 dimethyl-1H-pyrrolo[3,2- d]pyrimidine-2,4(3H,5H)-dione2160 P06 Oc1ccc2c(═O)cc(oc2c1O) 7,8-dihydroxy-2-phenyl-4H- 54.25 0.00c3ccccc3 chromen-4-one 1398 P09 Cc1cc(O)cc(O)c1C(═O)1-(2,4-dihydroxy-6-methylphenyl)-2- 54.25 0.00 COc2ccccc2phenoxyethanone

1. A method of treating or preventing a disease caused by a Plasmodiumor Theileria parasite in an individual in need thereof, comprising thestep of inhibiting interaction of heme and Heme Detoxification Protein(HDP) in said individual.
 2. The method of claim 1, wherein said step ofinhibiting is carried out by administering to said individual one ormore compounds that inhibit interaction of heme and HDP.
 3. The methodof claim 2, wherein said one or more compounds bind to heme.
 4. Themethod of claim 3, wherein said one or more compounds prevent heme frombinding to HDP.
 5. The method of claim 3, wherein said one or morecompounds allow the binding of heme to HDP but prevent detoxification ofheme by HDP.
 6. The method of claim 2, wherein said one or morecompounds bind to HDP.
 7. The method of claim 6, wherein said one ormore compounds prevent binding of heme to HDP.
 8. The method of claim 6,wherein said one or more compounds prevent the production of hemozoinfrom bound heme.
 9. The method of claim 6, wherein said one or morecompounds bind at the active site of HDP.
 10. The method of claim 6,wherein said one or more compounds bind at an allosteric site of HDP.11. The method of claim 1, wherein said step of inhibiting is carriedout by modification of a cell membrane of said Plasmodium or Theileriaparasite.
 12. The method of claim 1, wherein said step of inhibiting iscarried out by inhibiting secretion of HDP from said Plasmodium orTheileria parasite.
 13. The method of claim 2 wherein said disease ismalaria.
 14. The method of claim 13, wherein said compound isadministered to said individual in combination with one or more of: anadditional antimalarial agent, an agent for reversing antimalarialresistance, and an adjuvant.
 15. The method of claim 14, wherein saidcompound is administered prior to, concurrent with, or subsequent toadministration of said additional antimalarial agent or said agent forreversing antimalarial resistance.
 16. The method of claim 14, whereinsaid additional antimalarial agent is selected from the group consistingof a) quinolines, b) folic acid antagonists, c) sulfonamides, and d)antibiotics.
 17. The method of claim 14, wherein said agent forreversing antimalarial resistance is an inhibitor of multidrugresistance.
 18. The method of claim 2, wherein said compound isadministered orally, parenterally, sublingually, rectally, topically orwith an inhalation spray.
 19. The method of claim 2 wherein said one ormore compounds is selected from the group consisting of:(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one;(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide;(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-6-ol;(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one;[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-ylethoxy)phenyl]methanone;1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H-benzimidazol-2-one;1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium;1H-perimidine-2-carboxylic acid;2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;2-(4-methoxyphenyl)pyridin-3-ol; 2-(morpholin-4-ylmethyl)-1-naphthol;2,2′-buta-1,3-diyne-1,4-diyldiphenol;2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium;2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dimethylquinolinium;2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol;2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2H)-dione;2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one;2-hydrazino-4-methylquinoline; 2-hydroxy-N-(4-propylbenzoyl)benzamide;3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phenol;3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propanamide;4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-indole-2-carbonitrile;4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4,4′-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;4,4′-methylenebis(3-hydroxy-2-naphthoicacid)-3,3′-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline(1:1); 4,4′-propane-2,2-diylbis(2-chlorophenol);4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]phenol;4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzene-1,3-diol;4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol-2-ylidene]cyclohexa-2,5-dien-1-one;4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;4-phenylquinolin-2-amine;5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-carboxamide;5-7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one;6-amino-1-ethylbenzo[cd]indol-2(1H)-one;6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carboxamide;7-chloro-N-phenylquinolin-4-amine;7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1; COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;ethyl1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxylate;ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;N-(2-ethoxyphenyl)-2-hydroxybenzamide;N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;N-(2-hydroxybenzoyl)-3-methoxybenzamide;N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;N-(3-furylcarbonyl)-2-hydroxybenzamide;N-(4-ethylbenzoyl)-2-hydroxybenzamide;N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;N-[(1S)-1-phenylethyl]quinazolin-4-amine;N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethanamine;N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;N-benzo[g]quinolin-4-yl-N′-isopropylbenzene-1,4-diamine;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;and O[C@H]1[C@H]2[C@H](CC(═O)O)C(═O)O[C@@H]3C(COC(═O)c4cc(O)c(O)c(O)c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O)c6cc(O)c(O)c(OC1═O)c26)[C@@H]3OC(═O)c7cc(O)c(O)c(O)c7.
 20. The method of claim 2 wherein said one ormore compounds is selected from the group consisting of:(10S)-10-(dimethylamino)-9-methyl-7H,10H-naphtho[1,8-gh]chromen-7-one;(1E,4E)-1-[4-(dimethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one;(1R,3R)-1-isopropyl-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylicacid;(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;(2E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenyl)acrylamide;(2E)-6-ethoxy-2-(2-hydroxybenzylidene)-1-benzothiophen-3(2H)-one;(2E)-N-(2-methyl-1,3-benzothiazol-6-yl)-3-phenylacrylamide;(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one;(2E,5Z)-2-[(2-chlorophenyl)imino]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-thiazolidin-4-one;(2R)-1-(benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol;(2R)-2-(2,4-dichlorophenoxy)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)propanamide;(2R)-2-[(5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-methylbutanoicacid;(2R)-2-[(E)-2-(1,3-benzodioxol-5-yl)vinyl]-5,6-dimethyl-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one;(2R,3Z)-6-chloro-3-[(dimethylamino)methylene]-2-methyl-2,3-dihydro-4H-thiochromen-4-one;(2S)-2-(4-chlorophenyl)-3-oxo-4-phenylbutanenitrile;(2S)-2-[(5E)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]succinicacid;(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide;(2S,3Z)-3-(1H-indol-3-ylmethylene)-2-phenyl-2,3-dihydro-4H-chromen-4-one;(2Z)-2-acetamido-N-(3,5-dimethylphenyl)-3-phenylacrylamide;(2Z,5E)-2-[(3,5-dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)-1,3-thiazolidin-4-one;(2Z,5Z)-2-[(2-chlorophenyl)imino]-5-(2-hydroxy-3-nitrobenzylidene)-1,3-thiazolidin-4-one;(3,5-dichloro-2-hydroxyphenyl)(isoxazol-4-yl)methanone;(3-{(E)-[1-(3-fluorophenyl)-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene]methyl}-1H-indol-1-yl)acetonitrile;(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-6-ol;(3aS,6aS)-3-benzoyl-1,5-diphenyl-3a,6a-dihydropyrrolo[3,4-c]pyrazole-4,6(1H,5H)-dione;(3R)-3-(2-hydroxy-4-methylphenyl)-N-(2-methoxyphenyl)-3-phenylpropanamide;(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one;(3R,3′R,4′S,6′R,8′S,8a′S)-5-(4-hydroxybut-1-yn-1-yl)-6′-[4-(2-hydroxyethoxy)phenyl]-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazine]-8′-carboxylic acid;(3S)-3-(2-hydroxy-4-methylbenzoyl)-2-(4-methylphenyl)isoindolin-1-one;(3S,3aR,6aR)-3-(5-bromo-2-hydroxyphenyl)-5-butyl-2-phenyldihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione;(3S,6S,7R,8aR)-3-(4-acetamidobutyl)-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazine-7-carboxylicacid;(3Z)-3-(3-hydroxy-4-methoxybenzylidene)-1-methyl-1,3-dihydro-2H-indol-2-one;(4E)-2-(4-methoxyphenyl)-4-[(4-methoxyphenyl)imino]-4H-chromen-6-ol;(4R)-3-(3,4-dichlorophenyl)-4-hydroxy-N-isopropyl-2-oxo-1,2,3,4-tetrahydroquinazoline-4-carboxamide;(4R)-4-(4-bromophenyl)-3-hydroxy-1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7(6H)-one;(4R)-4-(4-ethylphenyl)-3-hydroxy-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one;(4R)-5-(2-furylmethyl)-3-(2-hydroxyphenyl)-4-phenyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one;(4R)-N˜4˜-(6-chloro-2-methoxyacridin-9-yl)-N˜1˜,N˜1˜-diethylpentane-1,4-diamine;(4S)-4-(2-bromobenzoyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one;(4S)-4-(2-furyl)-3-hydroxy-7,7-dimethyl-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one;(4S)-5,7-dihydroxy-4-phenylchroman-2-one;(4S,5S)-3,5-diphenyl-4,5-dihydro-1H-pyrazol-4-ol;(4S,7R)-2-amino-4-isobutyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile;(4Z)-2-[2-(4-chlorophenoxy)pyridin-3-yl]-4-[(dimethylamino)methylene]-1,3-oxazol-5(4H)-one;(5E)-1-(4-methylpentyl)-5-(1H-pyrrol-2-ylmethylene)pyrimidine-2,4,6(1H,3H,5H)-trione;(5E)-3-allyl-5-(2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one;(5E)-5-[4-(diethylamino)benzylidene]-3-{[(2-methoxyphenyl)amino]methyl}-1,3-thiazolidine-2,4-dione;(5R)-5-methyl-4-phenyl-1,3,4-thiadiazolidine-2-thione;(5S,7R)-2,2-dimethyl-5,7-bis(2-phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3-d][1,3]dioxine;(5Z)-5-(4-hydroxybenzylidene)-3-[(2R)-tetrahydrofuran-2-ylmethyl]-2-thioxo-1,3-thiazolidin-4-one;(6E)-5-imino-6-{[1-(2-naphthyl)-1H-pyrrol-2-yl]methylene}-5,6-dihydro-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one;*c1ceccc1C2C(═O)N(C)c3ccccc3C2=O;[(2R)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetic acid;[(5E)-4-oxo-5-(3-thienylmethylene)-2-thioxo-1,3-thiazolidin-3-yl]aceticacid;[(5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]aceticacid;[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-ylethoxy)phenyl]methanone;{4-[(4-methylphenyl)sulfonyl]phenyl}hydrazine;1-(2,4-dihydroxy-6-methylphenyl)-2-phenoxyethanone;1-(2,4-dihydroxyphenyl)-2-(4-isopropylphenoxy)ethanone;1-(3,4-dihydroxyphenyl)-2-({4-[(3,5-dimethoxyphenyl)amino]quinazolin-2-yl}thio)ethanone;1-(4-chlorophenyl)-1-hydroxy-3-phenylurea;1-(4-hydroxy-3,5-dimethylphenyl)-2-[(4-methylphenyl)thio]ethanone;1-(4-iodo-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-ol;1-(5-butyl-2,4-dihydroxyphenyl)-2-pyridin-2-ylethanone;1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)ethanone;1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;1-[(2S)-³-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H-benzimidazol-2-one;1-[4-(7-chloroquinolin-4-yl)piperazino]propan-1-one;1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium;1-ethynyl-2-phenoxybenzene; 1H-perimidine-2-carboxylic acid;2-(1,3-benzodioxol-5-yl)-1-(2,4-dihydroxy-5-propylphenyl)ethanone;2-(1H-benzimidazol-1-yl)-1-(5-ethyl-2,4-dihydroxyphenyl)ethanone;2-(2,4-dichlorophenyl)-1H-imidazo[4,5-b]pyridin-1-ol;2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one;2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one;2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;2-(4-chlorophenyl)-4H-1,3-benzoxazin-4-one;2-(4-chlorophenyl)-5-{[(4-pyridin-3-ylpyrimidin-2-yl)thio]methyl}-2,4-dihydro-3H-pyrazol-3-one;2-(4-fluoro-3-phenoxyphenyl)-3-hydroxy-4H-chromen-4-one;2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-N-(4-methylphenyl)acetamide;2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;2-(4-methoxyphenyl)pyridin-3-ol;2-(4-methylphenyl)-4H-1,3-benzoxazin-4-one;2-(morpholin-4-ylmethyl)-1-naphthol;2,2′-buta-1,3-diyne-1,4-diyldiphenol; 2,2′-thiobis(4-chlorophenol);2,4-dichloro-1-naphthyl[2,2,2-trifluoro-1-methyl-1-(trifluoromethyl)ethyl]carbamate;2-[(2-phenoxyethyl)thio]quinazoline-4-thiol;2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-ethylacetamide;2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]-N-1-naphthylacetamide;2-[(5S)-1-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl]phenol;2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium;2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dimethylquinolinium;2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol;2-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol;2-[5-(ethylsulfonyl)-2-hydroxyphenyl]-1H-benzo[de]isoquinoline-1,3(2H)-dione;2-{(1R)-1-[(1-allyl-1H-benzimidazol-2-yl)amino]ethyl}-4-chlorophenol;2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2H)-dione;2-{[5-chloro-6-methyl-2-(2-pyridinyl)-4-pyrimidinyl]sulfanyl}-1-phenyl-1-ethanone;2-amino-1-(2,4-dimethylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carbonitrile;2-amino-5-butyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenylnicotinonitrile;2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one;2-anilino-2-oxoethyl 2-(4-chlorobenzoyl)benzoate;2-chloro-5-phenyl-3-pyridin-4-yl-4H-1,4-thiazine;2-chloro-8-hydroxy-10,10-dimethyl-7-phenylpyrido[1,2-a]indol-6(10H)-one;2-hydrazino-4,6-diphenylpyrimidine; 2-hydrazino-4-methylquinoline;2-hydroxy-N-(4-methylphenyl)benzamide;2-hydroxy-N-(4-propylbenzoyl)benzamide;2-hydroxy-N-[(4-methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]benzamide;2-hydroxy-N-[2-(2-methoxyphenyl)acetyl]benzamide;2-hydroxy-N-{[2-(4-methylphenoxy)-3-pyridinyl]carbonyl}benzamide;2-hydroxy-N-pyridin-3-ylbenzamide; 2-phenyl-4H-thiochromene-4-thione;2-phenyl-5-({[5-(trifluoromethyl)pyridin-2-yl]sulfonyl}methyl)-2,4-dihydro-3H-pyrazol-3-one;2-phenyl-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one;3-(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-N-(2-hydroxyphenyl)-N-(4-methoxybenzyl)propanamide;3-(1-acetyl-1H-indol-3-yl)-4-hydroxy-2H-chromen-2-one;3-(1H-benzimidazol-1-yl)-6-ethyl-7-hydroxy-4H-chromen-4-one;3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-(3-{[(4-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-(4-bromophenyl)-7-hydroxy-2-methyl-4H-chromen-4-one;3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one;3-(5-{(Z)-[5-(4-methylphenyl)-2-oxofuran-3(2H)-ylidene]methyl}-2-furyl)benzoicacid; 3-(quinazolin-4-ylamino)phenyl thiophene-2-carboxylate;3,4-dimethoxy-N-(4-methyl-1,3-benzothiazol-2-yl)benzamide;3,5-dichloro-2-hydroxybenzaldehydeN-tert-butyl-N′-methylthiosemicarbazone;3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phenol;3-[2-(4-methoxyphenyl)ethyl]-10-methyl-6-phenyl-3,4-dihydro-2H,8H-chromeno[6,7-e][1,3]oxazin-8-one;3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propanamide;3-{2-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-1H-pyrrol-1-yl}benzoicacid; 3-benzyl-4-hydroxy-1,2-dihydroquinolin-2-one;3-benzyl-4-hydroxy-1-phenylquinolin-2(1H)-one;3-benzyl-5,6-bis(4-methoxyphenyl)furo[2,3-d]pyrimidin-4(3H)-imine;3-benzyl-5-ethyl-4-hydroxy-6-phenyl-1-(1,3-thiazol-2-yl)pyridin-2(1H)-one;3-benzyl-6-ethoxy-4-hydroxyquinolin-2(1H)-one;3-chloro-N-[2-(methylthio)-1,3-benzothiazol-6-yl]benzamide;3-hydroxy-N-(2-methylphenyl)-2-naphthamide;3-methoxy-2-methyl-6-[1′-phenyl-5-(trifluoromethyl)-1H,1′H-4,4′-bipyrazol-3-yl]phenol;3-methoxy-N-(3-[1,3]oxazolo[4,5-b]pyridin-2-ylphenyl)benzamide;3-methyl-1-[3-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol;3-methyl-1-phenyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]pyridin-6-one;3-methyl-4-[(4-methylphenyl)thio]-1-phenyl-1H-pyrazol-5-ylmethoxyacetate;3-oxo-3-[3-({[3-(trifluoromethyl)phenyl]thio}methyl)phenyl]propanenitrile;4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-indole-2-carbonitrile;4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;4-(1-propyl-1H-benzimidazol-2-yl)aniline;4-(2,6-dichlorobenzyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol;4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4,4′-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;4,4′-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(oxy)]diphenol;4,4′-methylenebis(3-hydroxy-2-naphthoicacid)-3,3′-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline(1:1); 4,4′-propane-2,2-diylbis(2-chlorophenol);4-[(3S,6S,7R,8aR)-7-{[2-(4-{(3S,3′S,4′R,6′R,8′R,8a′R)-8′-[(allyloxy)carbonyl]-5-iodo-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazin]-6′-yl}phenoxy)ethoxy]carbonyl}-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trimethylbutan-1-aminium;4-[(4-chlorophenyl)thio]-3-methyl-1-phenyl-1H-pyrazol-5-ol;4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]phenol;4-[1-(4-hydroxy-3-methoxybenzyl)-1H-benzimidazol-2-yl]-2-methoxyphenol;4-[1′-phenyl-5-(trifluoromethyl)-1H,1′H-4,4′-bipyrazol-3-yl]benzene-1,3-diol;4-[4-(1,3-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]benzene-1,3-diol;4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzene-1,3-diol;4-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrazol-5-amine;4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol-2-ylidene]cyclohexa-2,5-dien-1-one;4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en-1-yl]amino}benzoic acid;4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;4-{[(2S)-2-ethylpiperidin-1-yl]methyl}-3-hydroxy-1-methyl-6H-benzo[c]chromen-6-one;4-bromo-2-[(E)-(4H-1,2,4-triazol-4-ylimino)methyl]phenol;4-bromo-2-[5-(2-furyl)-1H-pyrazol-3-yl]phenol;4-bromo-6-chloro-2-oxo-1,3-benzoxathiol-5-yl ethyl carbonate;4-ethyl-6-[4-(1-methyl-1H-benzimidazol-2-yl)-1H-pyrazol-3-yl]benzene-1,3-diol;4-fluoro-N-[3-(trifluoromethyl)phenyl]benzamide;4-hydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-2-one;4-hydroxy-3-propylquinolin-2(1H)-one;4-hydroxy-5-phenyl-6H-pyrido[3,2,1-jk]carbazol-6-one;4-hydroxy-8-methyl-3-{(E)-[(3R)-5-oxo-1,3-diphenylpyrazolidin-4-ylidene]methyl}quinolin-2(1H)-one; 4-phenylquinolin-2-amine;5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-bromo-2-hydroxybenzoyl)-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-carboxamide;5-(benzoylamino)-N,N′-bis(2-hydroxyphenyl)isophthalamide;5-(diethylamino)-2-{(E)-[(2-phenylethyl)imino]methyl}phenol;5,7-dihydroxy-4-propyl-2H-chromen-2-one;5-[(4-methylphenyl)thio]quinazoline-2,4-diamine;5-{2-[(3,4-dichlorophenyl)thio]ethyl}-2-methylpyridine;5-benzyl-3-phenyl-5H-pyrazolo[4,3-c]quinolines;5-benzyl-4-hydroxy-6H-pyrido[3,2,1-jk]carbazol-6-one;5-chloro-2-hydroxy-N-phenylbenzamide;5-hydroxy-4-methyl-7-propyl-2H-chromen-2-one;5-methoxy-2-[3-methyl-4-(1,3-thiazol-4-yl)isoxazol-5-yl]phenol;5-methyl-2-[5-(2-thienyl)-1H-pyrazol-3-yl]phenol;6-(4-chlorophenyl)-7-hydroxy-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione″6,6′-biquinoline;6-[(S)-[4-(dimethylamino)phenyl](piperidin-1-yl)methyl]-1,3-benzodioxol-5-ol;6-{[(2-ethylphenyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;6-{[(4-chlorophenyl)thio]methyl}-2-phenyl-1H-pyrazolo[3,4-b]pyridine-3,4(2H,7H)-dione;6-{[(4-ethoxyphenyl)(methyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;6-allyl-7-hydroxy-4,8-dimethyl-2H-chromen-2-one;6-amino-1-ethylbenzo[cd]indol-2(1H)-one;6-benzyl-7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one;6-bromo-2-(trifluoromethyl)quinolin-4-ol;6-butyl-2-(2-furyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-7-one;6-chloro-2-(4-chlorophenyl)-1H-benzimidazol-1-ol;6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide;6-chloro-3-(4-methylphenyl)-3,4-dihydro-2H-1,3-benzoxazine;6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;6-ethyl-7-hydroxy-3-(1-methyl-1H-benzimidazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one;6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one;6-methoxy-N-[(1S)-1-methylpropyl]furo[2,3-b]quinoline-2-carboxamide;6-phenyl[1,2,3,4]tetraazolo[1,5-b]pyridazin-7-ol;7-(4-bromophenyl)-5-hydroxy-1,3-benzoxathiol-2-one;7,8-dihydroxy-2-phenyl-4H-chromen-4-one;7,8-dihydroxy-4-phenyl-2H-chromen-2-one;7-[(2E)-2-(4-fluoro-3-phenoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-[2-chloro-5-(trifluoromethyl)phenyl]-5-hydroxy-1,3-benzoxathiol-2-one;7-{(2E)-2-[(2-fluorobiphenyl-4-yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-benzyl-8-hydroxy-10,10-dimethyl-6,10-dihydropyrido[1,2-a]indol-6-one;7-chloro-4-piperidinoquinoline;7-chloro-N-(3-fluoro-4-methylphenyl)quinolin-4-amine;7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carboxamide;7-chloro-N-phenylquinolin-4-amine;7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;7-hydroxy-5-methyl-3-(1-phenyl-1H-pyrazol-4-yl)-2-(trifluoromethyl)-4H-chromen-4-one;7-hydroxy-6-methyl-3-(4-methyl-1,3-thiazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one;8-(trifluoromethoxy)-2-(trifluoromethyl)quinolin-4-ol;8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;8-[(2E)-2-(5-bromo-2-methoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;8-{(2E)-2-[(6-bromo-1,3-benzodioxol-5-yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-8-oxooctanamide;8-methoxy-N,N-dimethyl-5H-pyrimido[5,4-b]indol-4-amine;allyl(3R,3′R,4′S,6′R,8′S,8a′S)-6′-{4-[2-({[(3S,6R,7S,8aS)-3-[4-(dimethylamino)butyl]-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7-yl]carbonyl}oxy)ethoxy]phenyl}-5-iodo-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazine]-8′-carboxylate”bis[4-(dimethylamino)phenyl]methanone oxime;CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;COC(═O)[C@]1(Cc2ccc(O)c(CC═C(C)C)c2)OC(═O)C(═C1c3ccc(O)cc3)O;COc1cc(/C═C/2\Oc3cc(O)ccc3C2=O)ccc1O;COc1cc(ccc1O)c2oc3cc(O)cc(O)c3c(═O)c2O;COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;ethyl(2E)-3-(2-hydroxy-5-nitrophenyl)acrylate; ethyl1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxylate;ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate; ethyl4-(benzylamino)-6-ethoxyquinoline-3-carboxylate; ethyl4-[({[(5R)-5-ethyl-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-2-yl]thio}acetyl)amino]benzoate;ethyl 4-[(2-phenylethyl)amino]quinoline-3-carboxylate; ethyl4-{[(2-anilino-2-oxoethyl)thio]methyl}-5-hydroxy-2-phenyl-1-benzofuran-3-carboxylate;ethyl 4-{[(2E)-3-(2-thienyl)prop-2-enoyl]amino}benzoate; ethyl6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-{[(4-methylphenyl)thio]methyl}-1H-indole-3-carboxylate;ethyl 6-ethoxy-4-{[(1R)-1-methylpropyl]amino}quinoline-3-carboxylate;ethyl 6-methyl-4-[(4-morpholin-4-ylphenyl)amino]quinoline-3-carboxylate;isopropyl(2S)-2-{[(2S)-2-{[(2S,3R)-2-{[(2S)-2-amino-3-mercaptopropyl]amino}-3-methylpentyl]oxy}-3-phenylpropanoyl]amino}-4-(methylsulfonyl)butanoate;methyl(2Z)-2-(4-hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5-carboxylate;methyl 1-hydroxy-3-methylpyrido[1,2-a]benzimidazole-4-carboxylate;methyl2,3-bis-O-(biphenyl-2-ylcarbamoyl)-4-O-[(3-ethylphenyl)carbamoyl]-alpha-L-idopyranoside;methyl5-hydroxy-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate;N-(1,3-benzodioxol-5-yl)-7-chloroquinolin-4-amine;N-(2,3-dihydro-1-benzofuran-5-ylcarbonyl)-2-hydroxybenzamide;N-(2,5-dimethylphenyl)benzamide;N-(2-chlorobenzyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;N-(2-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-sulfonamide;N-(2-ethoxyphenyl)-2-hydroxybenzamide;N-(2-hydroxy-4-methylphenyl)-4-[(methylthio)methyl]benzamide;N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;N-(2-hydroxybenzoyl)-3-methoxybenzamide;N-(2-hydroxybenzoyl)-4-(trifluoromethyl)benzamide;N-(2-hydroxyphenyl)-8-[(2E)-2-(1-naphthylmethylene)hydrazino]-8-oxooctanamide;N-(3-bromo-4-hydroxy-1-naphthyl)-4-chlorobenzenesulfonamide;N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;N-(3-chlorophenyl)-4-(5-hydroxy-1-phenyl-1H-pyrazol-3-yl)piperidine-1-carbothioamide;N-(3-furylcarbonyl)-2-hydroxybenzamide;N-(3-hydroxypyridin-2-yl)-4-phenoxybenzamide;N-(3-imidazo[1,2-a]pyrimidin-2-ylphenyl)cyclopentanecarboxamide;N-(4-bromophenyl)-2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]acetamide;N-(4-carbamoylphenyl)-1-phenyl-3-(2-thienyl)-1H-pyrazole-4-carboxamide;N-(4-ethylbenzoyl)-2-hydroxybenzamide;N-(4-fluorophenyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;N-(5-{[(1S)-1-methylpropyl]thio}-1,3,4-thiadiazol-2-yl)-2-(trifluoromethyl)benzamide;N-(5-hydroxy-1-naphthyl)-4-methylbenzenesulfonamide;N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;N-(6-methyl-1,3-benzothiazol-2(3H)-ylidene)thiophene-2-carboxamide;N-(cyclohexylcarbonyl)-2-hydroxybenzamide;N,2-diphenylquinazolin-4-amine;N,N,8-trimethyl-5H-pyrimido[5,4-b]indol-4-amine;N,N′-1H-isoindole-1,3(2H)-diylidenedianiline;N,N-diethyl-8-methyl-5H-pyrimido[5,4-b]indol-4-amine;N,N-dimethyl-4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzenesulfonamide;N-[(1E)-(9-ethyl-9H-carbazol-3-yl)methylene]-4H-1,2,4-triazol-4-amine;N-[(1E)-1H-indol-3-ylmethylene]-1-propyl-1H-benzimidazol-2-amine;N-[(1S)-1-benzylpropyl]-6-[(4-methylpiperidin-1-yl)sulfonyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide;N-[(1S)-1-phenylethyl]quinazolin-4-amine;N-[(3-chloro-1-benzothiophen-2-yl)carbonyl]-2-hydroxybenzamide;N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethanamine;N-[2-(1H-benzimidazol-2-yl)phenyl]-2-methylpropanamide;N-[2-chloro-5-(trifluoromethyl)phenyl]-2-(4,4-dimethyl-2,6-dioxocyclohexyl)acetamide;N-[2-hydroxy-3-(4-oxo-4H-chromen-2-yl)phenyl]acetamide;N-[3-(1,3-benzothiazol-2-yl)-4-hydroxyphenyl]-2,2-dimethylpropanamide;N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]benzenesulfonamide;N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]thiophene-2-sulfonamide;N-[4-(1H-benzimidazol-2-yl)phenyl]-2-(2-methoxyphenyl)acetamide;N-[4-(ethylsulfonyl)-2-hydroxyphenyl]benzamide;N-[5-(ethylsulfonyl)-2-hydroxyphenyl]-2-(4-methoxyphenoxy)acetamide;N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;N-benzo[g]quinolin-4-yl-N′-isopropylbenzene-1,4-diamine;N-benzyl-2-hydroxybenzamide;N-benzyl-6-{[(3-methoxyphenyl)amino]sulfonyl}-N-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide;N-ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)/C═C/c4ccccc4)c(O)c3)[C@@H]1O;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O)cc(O)c4C3=O)c5ccc(O)cc5)c(O)cc(O)c2C1=O)c6ccc(O)cc6;O[C@H]1[C@H]2[C@H](CC(═O)O)C(═O)O[C@@H]3C(COC(═O)c4cc(O)c(O)c(O)c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O)c6cc(O)c(O)c(OC1=O)c26)[C@@H]3OC(═O)c7cc(O)c(O)c(O)c7;OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O)cc(O)c4c3=O)c5ccc(O)cc5)[C@H](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O; andOC1[C@H](OC(═O)c2cc(O)c(O)c(O)c2)OC3COC(═O)c4cc(O)c(O)c(O)c4-c5c(O)c(O)c(O)cc5C(═O)O[C@@H]1[C@@H]3OC(═O)c6cc(O)c(O)c(O)c6c7c(O)c(O)c(O)cc7C(═O)OOc1ccc(cc1)[C@H]2CC(═O)c3c(O)cc(O)c([C@H]4[C@@H](Oc5cc(O)cc(O)c5C4=O)c6ccc(O)cc6)c3O2Oc1ccc2C(═O)/C(═C/c3ccc(O)c(O)c3)/Oc2c1.
 21. A method of inhibitingheme detoxification in a Plasmodium or Theileria parasite, comprisingthe step of preventing or attenuating the production of hemozoin by HDPin said Plasmodium or Theileria parasite.
 22. The method of claim 21wherein said step of preventing or attenuating is carried out by aprocess selected from the group consisting of: 1) inhibiting interactionof heme and HDP; 2) preventing an interaction of HDP or heme withcofactors; 3) preventing dimerization of HDP; and 4) preventinginteraction of HDP or heme with lipids.
 23. The method of claim 22,wherein said cofactors are selected from the group consisting of metalions, natural ligands or protein factors.
 24. The method of claim 22,wherein said step of preventing or attenuating is carried out byadministering to said individual one or more compounds selected from thegroup consisting of:(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one;(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide;(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-6-ol;(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one;[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-ylethoxy)phenyl]methanone;1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H-benzimidazol-2-one;1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium;1H-perimidine-2-carboxylic acid;2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;2-(4-methoxyphenyl)pyridin-3-ol; 2-(morpholin-4-ylmethyl)-1-naphthol;2,2′-buta-1,3-diyne-1,4-diyldiphenol;2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium;2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dimethylquinolinium;2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol;2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2H)-dione;2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one;2-hydrazino-4-methylquinoline; 2-hydroxy-N-(4-propylbenzoyl)benzamide;3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phenol;3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propanamide;4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-indole-2-carbonitrile;4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4,4′-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;4,4′-methylenebis(3-hydroxy-2-naphthoicacid)-3,3′-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline(1:1); 4,4′-propane-2,2-diylbis(2-chlorophenol);4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]phenol;4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzene-1,3-diol;4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol-2-ylidene]cyclohexa-2,5-dien-1-one;4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;4-phenylquinolin-2-amine;5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-carboxamide;5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one;6-amino-1-ethylbenzo[cd]indol-2(1H)-one;6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carboxamide;7-chloro-N-phenylquinolin-4-amine;7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1; COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;ethyl1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxylate;ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;N-(2-ethoxyphenyl)-2-hydroxybenzamide;N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;N-(2-hydroxybenzoyl)-3-methoxybenzamide;N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;N-(3-furylcarbonyl)-2-hydroxybenzamide;N-(4-ethylbenzoyl)-2-hydroxybenzamide;N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;N-[(1S)-1-phenylethyl]quinazolin-4-amine;N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethanamine;N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;N-benzo[g]quinolin-4-yl-N′-isopropylbenzene-1,4-diamine;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;and O[C@H]1[C@H]2[C@H](CC(═O)O)C(═O)O[C@@H]3C(COC(═O)c4cc(O)c(O)c(O)c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O)c6cc(O)c(O)c(OC1=O)c26)[C@@H]3OC(═O)c7cc(O)c(O)c(O)c7.25. The method of claim 21 wherein said step of preventing orattenuating is carried out by administering to said individual one ormore compounds selected from the group consisting of:(10S)-10-(dimethylamino)-9-methyl-7H,10H-naphtho[1,8-gh]chromen-7-one;(1E,4E)-1-[4-(dimethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one;(1R,3R)-1-isopropyl-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylicacid;(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;(2E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenyl)acrylamide;(2E)-6-ethoxy-2-(2-hydroxybenzylidene)-1-benzothiophen-3(2H)-one;(2E)-N-(2-methyl-1,3-benzothiazol-6-yl)-3-phenylacrylamide;(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one;(2E,5Z)-2-[(2-chlorophenyl)imino]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-thiazolidin-4-one;(2R)-1-(benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol;(2R)-2-(2,4-dichlorophenoxy)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)propanamide;(2R)-2-[(5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-methylbutanoicacid;(2R)-2-[(E)-2-(1,3-benzodioxol-5-yl)vinyl]-5,6-dimethyl-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one;(2R,3Z)-6-chloro-3-[(dimethylamino)methylene]-2-methyl-2,3-dihydro-4H-thiochromen-4-one;(2S)-2-(4-chlorophenyl)-3-oxo-4-phenylbutanenitrile;(2S)-2-[(5E)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]succinicacid;(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide;(2S,3Z)-3-(1H-indol-3-ylmethylene)-2-phenyl-2,3-dihydro-4H-chromen-4-one;(2Z)-2-acetamido-N-(3,5-dimethylphenyl)-3-phenylacrylamide;(2Z,5E)-2-[(3,5-dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)-1,3-thiazolidin-4-one;(2Z,5Z)-2-[(2-chlorophenyl)imino]-5-(2-hydroxy-3-nitrobenzylidene)-1,3-thiazolidin-4-one;(3,5-dichloro-2-hydroxyphenyl)(isoxazol-4-yl)methanone;(3-{(E)-[1-(3-fluorophenyl)-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene]methyl}-1H-indol-1-yl)acetonitrile;(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-6-ol;(3aS,6aS)-3-benzoyl-1,5-diphenyl-3a,6a-dihydropyrrolo[3,4-c]pyrazole-4,6(1H,5H)-dione;(3R)-3-(2-hydroxy-4-methylphenyl)-N-(2-methoxyphenyl)-3-phenylpropanamide;(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one;(3R,3′R,4′S,6′R,8′S,8a′S)-5-(4-hydroxybut-1-yn-1-yl)-6′-[4-(2-hydroxyethoxy)phenyl]-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazine]-8′-carboxylicacid;(3S)-3-(2-hydroxy-4-methylbenzoyl)-2-(4-methylphenyl)isoindolin-1-one;(3S,3aR,6aR)-3-(5-bromo-2-hydroxyphenyl)-5-butyl-2-phenyldihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione;(3S,6S,7R,8aR)-3-(4-acetamidobutyl)-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazine-7-carboxylicacid;(3Z)-3-(3-hydroxy-4-methoxybenzylidene)-1-methyl-1,3-dihydro-2H-indol-2-one;(4E)-2-(4-methoxyphenyl)-4-[(4-methoxyphenyl)imino]-4H-chromen-6-ol;(4R)-3-(3,4-dichlorophenyl)-4-hydroxy-N-isopropyl-2-oxo-1,2,3,4-tetrahydroquinazoline-4-carboxamide;(4R)-4-(4-bromophenyl)-3-hydroxy-1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7(6H)-one;(4R)-4-(4-ethylphenyl)-3-hydroxy-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one;(4R)-5-(2-furylmethyl)-3-(2-hydroxyphenyl)-4-phenyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one;(4R)-N˜4˜-(6-chloro-2-methoxyacridin-9-yl)-N˜1˜,N˜1˜-diethylpentane-1,4-diamine;(4S)-4-(2-bromobenzoyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one;(4S)-4-(2-furyl)-3-hydroxy-7,7-dimethyl-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one;(4S)-5,7-dihydroxy-4-phenylchroman-2-one;(4S,5S)-3,5-diphenyl-4,5-dihydro-1H-pyrazol-4-ol;(4S,7R)-2-amino-4-isobutyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile;(4Z)-2-[2-(4-chlorophenoxy)pyridin-3-yl]-4-[(dimethylamino)methylene]-1,3-oxazol-5(4H)-one;(5E)-1-(4-methylpentyl)-5-(1H-pyrrol-2-ylmethylene)pyrimidine-2,4,6(1H,3H,5H)-trione;(5E)-3-allyl-5-(2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one;(5E)-5-[4-(diethylam-ino)benzylidene]-3-{[(2-methoxyphenyl)amino]methyl}1,3-thiazolidine-2,4-dione;(5R)-5-methyl-4-phenyl-1,3,4-thiadiazolidine-2-thione;(5S,7R)-2,2-dimethyl-5,7-bis(2-phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3-d][1,3]dioxine;(5Z)-5-(4-hydroxybenzylidene)-3-[(2R)-tetrahydrofuran-2-ylmethyl]-2-thioxo-1,3-thiazolidin-4-one;(6E)-5-imino-6-{[1-(2-naphthyl)-1H-pyrrol-2-yl]methylene}-5,6-dihydro-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one;*cccccc1C2C(═O)N(C)c3ccccc3C2=O;[(2R)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetic acid;[(5E)-4-oxo-5-(3-thienylmethylene)-2-thioxo-1,3-thiazolidin-3-yl]aceticacid;[(5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]aceticacid;[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-ylethoxy)phenyl]methanone;{4-[(4-methylphenyl)sulfonyl]phenyl}hydrazine;1-(2,4-dihydroxy-6-methylphenyl)-2-phenoxyethanone;1-(2,4-dihydroxyphenyl)-2-(4-isopropylphenoxy)ethanone;1-(3,4-dihydroxyphenyl)-2-({4-[(3,5-dimethoxyphenyl)amino]quinazolin-2-yl}thio)ethanone;1-(4-chlorophenyl)-1-hydroxy-3-phenylurea;1-(4-hydroxy-3,5-dimethylphenyl)-2-[(4-methylphenyl)thio]ethanone;1-(4-iodo-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-ol;1-(5-butyl-2,4-dihydroxyphenyl)-2-pyridin-2-ylethanone;1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)ethanone;1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H-benzimidazol-2-one;1-[4-(7-chloroquinolin-4-yl)piperazino]propan-1-one;1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium;1-ethynyl-2-phenoxybenzene; 1H-perimidine-2-carboxylic acid;2-(1,3-benzodioxol-5-yl)-1-(2,4-dihydroxy-5-propylphenyl)ethanone;2-(1H-benzimidazol-1-yl)-1-(5-ethyl-2,4-dihydroxyphenyl)ethanone;2-(2,4-dichlorophenyl)-1H-imidazo[4,5-b]pyridin-1-ol;2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one;2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one;2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;2-(4-chlorophenyl)-4H-1,3-benzoxazin-4-one;2-(4-chlorophenyl)-5-{[(4-pyridin-3-ylpyrimidin-2-yl)thio]methyl}-2,4-dihydro-3H-pyrazol-3-one;2-(4-fluoro-3-phenoxyphenyl)-3-hydroxy-4H-chromen-4-one;2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-N-(4-methylphenyl)acetamide;2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;2-(4-methoxyphenyl)pyridin-3-ol;2-(4-methylphenyl)-4H-1,3-benzoxazin-4-one;2-(morpholin-4-ylmethyl)-1-naphthol;2,2′-buta-1,3-diyne-1,4-diyldiphenol; 2,2′-thiobis(4-chlorophenol);2,4-dichloro-1-naphthyl[2,2,2-trifluoro-1-methyl-1-(trifluoromethyl)ethyl]carbamate;2-[(2-phenoxyethyl)thio]quinazoline-4-thiol;2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-ethylacetamide;2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]-N-1-naphthylacetamide;2-[(5S)-1-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl]phenol;2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium;2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dimethylquinolinium;2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol;2-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol;2-[5-(ethylsulfonyl)-2-hydroxyphenyl]-1H-benzo[de]isoquinoline-1,3(2H)-dione;2-{(1R)-1-[(1-allyl-1H-benzimidazol-2-yl)amino]ethyl}-4-chlorophenol;2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2H)-dione;2-{[5-chloro-6-methyl-2-(2-pyridinyl)-4-pyrimidinyl]sulfanyl}-1-phenyl-1-ethanone;2-amino-1-(2,4-dimethylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carbonitrile;2-amino-5-butyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenylnicotinonitrile;2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one;2-anilino-2-oxoethyl2-(4-chlorobenzoyl)benzoate;2-chloro-5-phenyl-3-pyridin-4-yl-4H-1,4-thiazine;2-chloro-8-hydroxy-10,10-dimethyl-7-phenylpyrido[1,2-a]indol-6(10H)-one;2-hydrazino-4,6-diphenylpyrimidine; 2-hydrazino-4-methylquinoline;2-hydroxy-N-(4-methylphenyl)benzamide;2-hydroxy-N-(4-propylbenzoyl)benzamide;2-hydroxy-N-[(4-methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]benzamide;2-hydroxy-N-[2-(2-methoxyphenyl)acetyl]benzamide;2-hydroxy-N-{[2-(4-methylphenoxy)-3-pyridinyl]carbonyl}benzamide;2-hydroxy-N-pyridin-3-ylbenzamide; 2-phenyl-4H-thiochromene-4-thione;2-phenyl-5-({[5-(trifluoromethyl)pyridin-2-yl]sulfonyl}methyl)-2,4-dihydro-3H-pyrazol-3-one;2-phenyl-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one;3-(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-N-(2-hydroxyphenyl)-N-(4-methoxybenzyl)propanamide;3-(1-acetyl-1H-indol-3-yl)-4-hydroxy-2H-chromen-2-one;3-(1H-benzimidazol-1-yl)-6-ethyl-7-hydroxy-4H-chromen-4-one;3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-(3-{[(4-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-(4-bromophenyl)-7-hydroxy-2-methyl-4H-chromen-4-one;3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one;3-(5-{(Z)-[5-(4-methylphenyl)-2-oxofuran-3(2H)-ylidene]methyl}-2-furyl)benzoicacid; 3-(quinazolin-4-ylamino)phenyl thiophene-2-carboxylate;3,4-dimethoxy-N-(4-methyl-1,3-benzothiazol-2-yl)benzamide;3,5-dichloro-2-hydroxybenzaldehydeN-tert-butyl-N′-methylthiosemicarbazone;3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phenol;3-[2-(4-methoxyphenyl)ethyl]-10-methyl-6-phenyl-3,4-dihydro-2H,8H-chromeno[6,7-e][1,3]oxazin-8-one;3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propanamide;3-{2-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-1H-pyrrol-1-yl}benzoicacid; 3-benzyl-4-hydroxy-1,2-dihydroquinolin-2-one;3-benzyl-4-hydroxy-1-phenylquinolin-2(1H)-one;3-benzyl-5,6-bis(4-methoxyphenyl)furo[2,3-d]pyrimidin-4(3H)-imine;3-benzyl-5-ethyl-4-hydroxy-6-phenyl-1-(1,3-thiazol-2-yl)pyridin-2(1H)-one;3-benzyl-6-ethoxy-4-hydroxyquinolin-2(1H)-one;3-chloro-N-[2-(methylthio)-1,3-benzothiazol-6-yl]benzamide;3-hydroxy-N-(2-methylphenyl)-2-naphthamide;3-methoxy-2-methyl-6-[1′-phenyl-5-(trifluoromethyl)-1H,1′H-4,4′-bipyrazol-3-yl]phenol;3-methoxy-N-(3-[1,3]oxazolo[4,5-b]pyridin-2-ylphenyl)benzamide;3-methyl-1-[3-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol;3-methyl-1-phenyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]pyridin-6-one;3-methyl-4-[(4-methylphenyl)thio]-1-phenyl-1H-pyrazol-5-ylmethoxyacetate;3-oxo-3-[3-({[3-(trifluoromethyl)phenyl]thio}methyl)phenyl]propanenitrile;4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-indole-2-carbonitrile;4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;4-(1-propyl-1H-benzimidazol-2-yl)aniline;4-(2,6-dichlorobenzyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol;4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4,4′-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;4,4′-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(oxy)]diphenol;4,4′-methylenebis(3-hydroxy-2-naphthoicacid)-3,3′-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline(1:1); 4,4′-propane-2,2-diylbis(2-chlorophenol);4-[(3S,6S,7R,8aR)-7-{[2-(4-{(3S,3′S,4′R,6′R,8′R,8a′R)-8′-[(allyloxy)carbonyl]-5-iodo-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazin]-6′-yl}phenoxy)ethoxy]carbonyl}-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trimethylbutan-1-aminium;4-[(4-chlorophenyl)thio]-3-methyl-1-phenyl-1H-pyrazol-5-ol;4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]phenol;4-[1-(4-hydroxy-3-methoxybenzyl)-1H-benzimidazol-2-yl]-2-methoxyphenol;4-[1′-phenyl-5-(trifluoromethyl)-1H,1′H-4,4′-bipyrazol-3-yl]benzene-1,3-diol;4-[4-(1,3-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]benzene-1,3-diol;4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzene-1,3-diol;4-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrazol-5-amine;4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol-2-ylidene]cyclohexa-2,5-dien-1-one;4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en-1-yl]amino}benzoic acid;4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;4-{[(2S)-2-ethylpiperidin-1-yl]methyl}-3-hydroxy-1-methyl-6H-benzo[c]chromen-6-one;4-bromo-2-[(E)-(4H-1,2,4-triazol-4-ylimino)methyl]phenol;4-bromo-2-[5-(2-furyl)-1H-pyrazol-3-yl]phenol;4-bromo-6-chloro-2-oxo-1,3-benzoxathiol-5-yl ethyl carbonate;4-ethyl-6-[4-(1-methyl-1H-benzimidazol-2-yl)-1H-pyrazol-3-yl]benzene-1,3-diol;4-fluoro-N-[3-(trifluoromethyl)phenyl]benzamide;4-hydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-2-one;4-hydroxy-3-propylquinolin-2(1H)-one;4-hydroxy-5-phenyl-6H-pyrido[3,2,1-jk]carbazol-6-one;4-hydroxy-8-methyl-3-{(E)-[(3R)-5-oxo-1,3-diphenylpyrazolidin-4-ylidene]methyl}quinolin-2(1H)-one; 4-phenylquinolin-2-amine;5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-bromo-2-hydroxybenzoyl)-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-carboxamide;5-(benzoylamino)-N,N′-bis(2-hydroxyphenyl)isophthalamide;5-(diethylamino)-2-{(E)-[(2-phenylethyl)imino]methyl}phenol;5,7-dihydroxy-4-propyl-2H-chromen-2-one;5-[(4-methylphenyl)thio]quinazoline-2,4-diamine;5-{2-[(3,4-dichlorophenyl)thio]ethyl}-2-methylpyridine;5-benzyl-3-phenyl-5H-pyrazolo[4,3-c]quinolines;5-benzyl-4-hydroxy-6H-pyrido[3,2,1-jk]carbazol-6-one;5-chloro-2-hydroxy-N-phenylbenzamide;5-hydroxy-4-methyl-7-propyl-2H-chromen-2-one;5-methoxy-2-[3-methyl-4-(1,3-thiazol-4-yl)isoxazol-5-yl]phenol;5-methyl-2-[5-(2-thienyl)-1H-pyrazol-3-yl]phenol;6-(4-chlorophenyl)-7-hydroxy-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione″6,6′-biquinoline;6-[(S)-[4-(dimethylamino)phenyl](piperidin-1-yl)methyl]-1,3-benzodioxol-5-ol;6-{[(2-ethylphenyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;6-{[(4-chlorophenyl)thio]methyl}-2-phenyl-1H-pyrazolo[3,4-b]pyridine-3,4(2H,7H)-dione;6-{[(4-ethoxyphenyl)(methyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;6-allyl-7-hydroxy-4,8-dimethyl-2H-chromen-2-one;6-amino-1-ethylbenzo[cd]indol-2(1H)-one;6-benzyl-7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one;6-bromo-2-(trifluoromethyl)quinolin-4-ol;6-butyl-2-(2-furyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-7-one;6-chloro-2-(4-chlorophenyl)-1H-benzimidazol-1-ol;6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide;6-chloro-3-(4-methylphenyl)-3,4-dihydro-2H-1,3-benzoxazine;6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;6-ethyl-7-hydroxy-3-(1-methyl-1H-benzimidazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one;6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one;6-methoxy-N-[(1S)-1-methylpropyl]furo[2,3-b]quinoline-2-carboxamide;6-phenyl[1,2,3,4]tetraazolo[1,5-b]pyridazin-7-ol;7-(4-bromophenyl)-5-hydroxy-1,3-benzoxathiol-2-one;7,8-dihydroxy-2-phenyl-4H-chromen-4-one;7,8-dihydroxy-4-phenyl-2H-chromen-2-one;7-[(2E)-2-(4-fluoro-3-phenoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-[2-chloro-5-(trifluoromethyl)phenyl]-5-hydroxy-1,3-benzoxathiol-2-one;7-{(2E)-2-[(2-fluorobiphenyl-4-yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-benzyl-8-hydroxy-10,10-dimethyl-6,10-dihydropyrido[1,2-a]indol-6-one;7-chloro-4-piperidinoquinoline;7-chloro-N-(3-fluoro-4-methylphenyl)quinolin-4-amine;7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carboxamide;7-chloro-N-phenylquinolin-4-amine;7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;7-hydroxy-5-methyl-3-(1-phenyl-1H-pyrazol-4-yl)-2-(trifluoromethyl)-4H-chromen-4-one;7-hydroxy-6-methyl-3-(4-methyl-1,3-thiazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one;8-(trifluoromethoxy)-2-(trifluoromethyl)quinolin-4-ol;8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;8-[(2E)-2-(5-bromo-2-methoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;8-{(2E)-2-[(6-bromo-1,3-benzodioxol-5-yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-8-oxooctanamide;8-methoxy-N,N-dimethyl-5H-pyrimido[5,4-b]indol-4-amine;allyl(3R,3′R,4′S,6′R,8′S,8a′S)-6′-{4-[2-({[(3S,6R,7S,8aS)-3-[4-(dimethylamino)butyl]-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7-yl]carbonyl}oxy)ethoxy]phenyl}-5-iodo-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazine]-8′-carboxylate″bis[4-(dimethylamino)phenyl]methanoneoxime; CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;COC(═O)[C@]1(Cc2ccc(O)c(CC═C(C)C)c2)OC(═O)C(═C1c3ccc(O)cc3)O;COc1cc(/C═C/2\Oc3cc(O)ccc3C2=O)ccc1O;COc1cc(ccc1O)c2oc3cc(O)cc(O)c3c(═O)c2O;COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;ethyl(2E)-3-(2-hydroxy-5-nitrophenyl)acrylate; ethyl1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxylate;ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate; ethyl4-(benzylamino)-6-ethoxyquinoline-3-carboxylate; ethyl4-[({[(5R)-5-ethyl-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-2-yl]thio}acetyl)amino]benzoate;ethyl 4-[(2-phenylethyl)amino]quinoline-3-carboxylate; ethyl4-{[(2-anilino-2-oxoethyl)thio]methyl}-5-hydroxy-2-phenyl-1-benzofuran-3-carboxylate;ethyl 4-{[(2E)-3-(2-thienyl)prop-2-enoyl]amino}benzoate; ethyl6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-{[(4-methylphenyl)thio]methyl}-1H-indole-3-carboxylate;ethyl 6-ethoxy-4-{[(1R)-1-methylpropyl]amino}quinoline-3-carboxylate;ethyl 6-methyl-4-[(4-morpholin-4-ylphenyl)amino]quinoline-3-carboxylate;isopropyl(2S)-2-{[(2S)-2-{[(2S,3R)-2-{[(2S)-2-amino-3-mercaptopropyl]amino}-3-methylpentyl]oxy}-3-phenylpropanoyl]amino}-4-(methylsulfonyl)butanoate;methyl(2Z)-2-(4-hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5-carboxylate;methyl 1-hydroxy-3-methylpyrido[1,2-a]benzimidazole-4-carboxylate;methyl2,3-bis-O-(biphenyl-2-ylcarbamoyl)-4-O-[(3-ethylphenyl)carbamoyl]-alpha-L-idopyranoside;methyl5-hydroxy-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate;N-(1,3-benzodioxol-5-yl)-7-chloroquinolin-4-amine;N-(2,3-dihydro-1-benzofuran-5-ylcarbonyl)-2-hydroxybenzamide;N-(2,5-dimethylphenyl)benzamide;N-(2-chlorobenzyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;N-(2-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-sulfonamide;N-(2-ethoxyphenyl)-2-hydroxybenzamide;N-(2-hydroxy-4-methylphenyl)-4-[(methylthio)methyl]benzamide;N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;N-(2-hydroxybenzoyl)-3-methoxybenzamide;N-(2-hydroxybenzoyl)-4-(trifluoromethyl)benzamide;N-(2-hydroxyphenyl)-8-[(2E)-2-(1-naphthylmethylene)hydrazino]-8-oxooctanamide;N-(3-bromo-4-hydroxy-1-naphthyl)-4-chlorobenzenesulfonamide;N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;N-(3-chlorophenyl)-4-(5-hydroxy-1-phenyl-1H-pyrazol-3-yl)piperidine-1-carbothioamide;N-(3-furylcarbonyl)-2-hydroxybenzamide;N-(3-hydroxypyridin-2-yl)-4-phenoxybenzamide;N-(3-imidazo[1,2-a]pyrimidin-2-ylphenyl)cyclopentanecarboxamide;N-(4-bromophenyl)-2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]acetamide;N-(4-carbamoylphenyl)-1-phenyl-3-(2-thienyl)-1H-pyrazole-4-carboxamide;N-(4-ethylbenzoyl)-2-hydroxybenzamide;N-(4-fluorophenyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;N-(5-{[(1S)-1-methylpropyl]thio}-1,3,4-thiadiazol-2-yl)-2-(trifluoromethyl)benzamide;N-(5-hydroxy-1-naphthyl)-4-methylbenzenesulfonamide;N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;N-(6-methyl-1,3-benzothiazol-2(3H)-ylidene)thiophene-2-carboxamide;N-(cyclohexylcarbonyl)-2-hydroxybenzamide;N,2-diphenylquinazolin-4-amine;N,N,8-trimethyl-5H-pyrimido[5,4-b]indol-4-amine;N,N′-1H-isoindole-1,3(2H)-diylidenedianiline;N,N-diethyl-8-methyl-5H-pyrimido[5,4-b]indol-4-amine;N,N-dimethyl-4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzenesulfonamide;N-[(1E)-(9-ethyl-9H-carbazol-3-yl)methylene]-4H-1,2,4-triazol-4-amine;N-[(1E)-1H-indol-3-ylmethylene]-1-propyl-1H-benzimidazol-2-amine;N-[(1S)-1-benzylpropyl]-6-[(4-methylpiperidin-1-yl)sulfonyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide;N-[(1S)-1-phenylethyl]quinazolin-4-amine;N-[(3-chloro-1-benzothiophen-2-yl)carbonyl]-2-hydroxybenzamide;N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethanamine;N-[2-(1H-benzimidazol-2-yl)phenyl]-2-methylpropanamide;N-[2-chloro-5-(trifluoromethyl)phenyl]-2-(4,4-dimethyl-2,6-dioxocyclohexyl)acetamide;N-[2-hydroxy-3-(4-oxo-4H-chromen-2-yl)phenyl]acetamide;N-[3-(1,3-benzothiazol-2-yl)-4-hydroxyphenyl]-2,2-dimethylpropanamide;N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]benzenesulfonamide;N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]thiophene-2-sulfonamide;N-[4-(1H-benzimidazol-2-yl)phenyl]-2-(2-methoxyphenyl)acetamide;N-[4-(ethylsulfonyl)-2-hydroxyphenyl]benzamide;N-[5-(ethylsulfonyl)-2-hydroxyphenyl]-2-(4-methoxyphenoxy)acetamide;N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;N-benzo[g]quinolin-4-yl-N′-isopropylbenzene-1,4-diamine;N-benzyl-2-hydroxybenzamide;N-benzyl-6-{[(3-methoxyphenyl)amino]sulfonyl}-N-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide;N-ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)/C═C/c4ccccc4)c(O)c3)[C@@H]1O;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3cCc(C(═O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O)cc(O)c4C3=O)c5ccc(O)cc5)c(O)cc(O)c2C1=O)c6ccc(O)cc6;O[C@H]1[C@H]2[C@H](CC(═O)O)C(═O)O[C@@H]3C(COC(═O)c4cc(O)c(O)c(O)c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O)c6cc(O)c(O)c(OC1=O)c26)[C@@H]3OC(═O)c7cc(O)c(O)c(O)c7;OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O)cc(O)c4c3=O)c5cCc(O)cc5)[C@H](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O;andOC1[C@H](OC(═O)c2cc(O)c(O)c(O)c2)OC3COC(═O)c4cc(O)c(O)c(O)c4-c5c(O)c(O)c(O)cc5C(═O)O[C@@H]1[C@@H]3OC(═O)c6cc(O)c(O)c(O)c6c7c(O)c(O)c(O)cc7C(═O)OOc1ccc(cc1)[C@H]2CC(═O)c3c(O)cc(O)c([C@H]4[C@@H](Oc5cc(O)cc(O)c5C4=O)c6ccc(O)cc6)c3O2Oc1ccc2C(═O)/C(═C/c3ccc(O)c(O)c3)/Oc2c1.
 26. The method of claim 21,wherein said method is used to treat or prevent malaria.
 27. A method oftreating an individual infected with Plasmodium or Theileria or who hasbeen or will be exposed to Plasmodium or Theileria, comprising the stepof providing said individual with one or more compounds that inhibit theability of HDP to produce hemozoin from heme.
 28. The method of claim27, wherein said one or more compounds bind to heme.
 29. The method ofclaim 28, wherein said one or more compounds prevent heme from bindingto HDP.
 30. The method of claim 28, wherein said one or more compoundsallow the binding of heme to HDP but prevent detoxification of heme byHDP.
 31. The method of claim 27, wherein said one or more compounds bindto HDP.
 32. The method of claim 31, wherein said one or more compoundsprevent binding of heme to HDP.
 33. The method of claim 31, wherein saidone or more compounds prevent the production of hemozoin from boundheme.
 34. The method of claim 31, wherein said one or more compound bindat the active site of HDP.
 35. The method of claim 31, wherein said oneor more compound bind at an allosteric site of HDP.
 36. The method ofclaim 27 wherein said one or more compounds is selected from the groupconsisting of:(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one;(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide;(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-6-ol;(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one;[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-ylethoxy)phenyl]methanone;1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H-benzimidazol-2-one;1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium;1H-perimidine-2-carboxylic acid;2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;2-(4-methoxyphenyl)pyridin-3-ol; 2-(morpholin-4-ylmethyl)-1-naphthol;2,2′-buta-1,3-diyne-1,4-diyldiphenol;2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium;2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dimethylquinolinium;2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol;2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2H)-dione;2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one;2-hydrazino-4-methylquinoline; 2-hydroxy-N-(4-propylbenzoyl)benzamide;3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-[(4-{[(2R)-tetrahydrofiuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phenol;3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propanamide;4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-indole-2-carbonitrile;4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4,4′-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;4,4′-methylenebis(3-hydroxy-2-naphthoicacid)-3,3′-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline(1:1); 4,4′-propane-2,2-diylbis(2-chlorophenol);4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]phenol;4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzene-1,3-diol;4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol-2-ylidene]cyclohexa-2,5-dien-1-one;4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;4-phenylquinolin-2-amine;5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-carboxamide;5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one;6-amino-1-ethylbenzo[cd]indol-2(1H)-one;6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carboxamide;7-chloro-N-phenylquinolin-4-amine;7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1; COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;ethyl1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxylate;ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;N-(2-ethoxyphenyl)-2-hydroxybenzamide;N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;N-(2-hydroxybenzoyl)-3-methoxybenzamide;N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;N-(3-furylcarbonyl)-2-hydroxybenzamide;N-(4-ethylbenzoyl)-2-hydroxybenzamide;N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;N-[(1S)-1-phenylethyl]quinazolin-4-amine;N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethanamine;N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;N-benzo[g]quinolin-4-yl-N′-isopropylbenzene-1,4-diamine;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;and O[C@H]1[C@H]2[C@H](CC(═O)O)C(═O)O[C@@H]3C(COC(═O)c4cc(O)c(O(O)O)c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O)c6cc(O)c(O)c(OC1=O)c26)[C@@H]3OC(═O)c7cc(O)c(O)c(O)c7.
 37. The method of claim 27 wherein said one ormore compounds is selected from the group consisting of(10S)-10-(dimethylamino)-9-methyl-7H,10H-naphtho[1,8-gh]chromen-7-one;(1E,4E)-1-[4-(dimethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one;(1R,3R)-1-isopropyl-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylicacid;(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;(2E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenyl)acrylamide;(2E)-6-ethoxy-2-(2-hydroxybenzylidene)-1-benzothiophen-3(2H)-one;(2E)-N-(2-methyl-1,3-benzothiazol-6-yl)-3-phenylacrylamide;(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one;(2E,5Z)-2-[(2-chlorophenyl)imino]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-thiazolidin-4-one;(2R)-1-(benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol;(2R)-2-(2,4-dichlorophenoxy)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)propanamide;(2R)-2-[(5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-methylbutanoicacid;(2R)-2-[(E)-2-(1,3-benzodioxol-5-yl)vinyl]-5,6-dimethyl-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one;(2R,3Z)-6-chloro-3-[(dimethylamino)methylene]-2-methyl-2,3-dihydro-4H-thiochromen-4-one;(2S)-2-(4-chlorophenyl)-3-oxo-4-phenylbutanenitrile;(2S)-2-[(5E)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]succinicacid;(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide;(2S,3Z)-3-(1H-indol-3-ylmethylene)-2-phenyl-2,3-dihydro-4H-chromen-4-one;(2Z)-2-acetamido-N-(3,5-dimethylphenyl)-3-phenylacrylamide;(2Z,5E)-2-[(3,5-dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)-1,3-thiazolidin-4-one;(2Z,5Z)-2-[(2-chlorophenyl)imino]-5-(2-hydroxy-3-nitrobenzylidene)-1,3-thiazolidin-4-one;(3,5-dichloro-2-hydroxyphenyl)(isoxazol-4-yl)methanone;(3-{(E)-[1-(3-fluorophenyl)-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene]methyl}-1H-indol-1-yl)acetonitrile;(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-6-ol;(3aS,6aS)-3-benzoyl-1,5-diphenyl-3a,6a-dihydropyrrolo[3,4-c]pyrazole-4,6(1H,5H)-dione;(3R)-3-(2-hydroxy-4-methylphenyl)-N-(2-methoxyphenyl)-3-phenylpropanamide;(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one;(3R,3′R,4′S,6′R,8′S,8a′S)-5-(4-hydroxybut-1-yn-1-yl)-6′-[4-(2-hydroxyethoxy)phenyl]-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazine]-8′-carboxylic acid;(3S)-3-(2-hydroxy-4-methylbenzoyl)-2-(4-methylphenyl)isoindolin-1-one;(3S,3aR,6aR)-3-(5-bromo-2-hydroxyphenyl)-5-butyl-2-phenyldihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione;(3S,6S,7R,8aR)-3-(4-acetamidobutyl)-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazine-7-carboxylicacid;(3Z)-3-(3-hydroxy-4-methoxybenzylidene)-1-methyl-1,3-dihydro-2H-indol-2-one;(4E)-2-(4-methoxyphenyl)-4-[(4-methoxyphenyl)imino]-4H-chromen-6-ol;(4R)-3-(3,4-dichlorophenyl)-4-hydroxy-N-isopropyl-2-oxo-1,2,3,4-tetrahydroquinazoline-4-carboxamide;(4R)-4-(4-bromophenyl)-3-hydroxy-1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7(6H)-one;(4R)-4-(4-ethylphenyl)-3-hydroxy-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one;(4R)-5-(2-furylmethyl)-3-(2-hydroxyphenyl)-4-phenyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one;(4R)-N˜4˜-(6-chloro-2-methoxyacridin-9-yl)-N˜1˜,N˜1˜-diethylpentane-1,4-diamine;(4S)-4-(2-bromobenzoyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one;(4S)-4-(2-furyl)-3-hydroxy-7,7-dimethyl-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one;(4S)-5,7-dihydroxy-4-phenylchroman-2-one;(4S,5S)-3,5-diphenyl-4,5-dihydro-1H-pyrazol-4-ol;(4S,7R)-2-amino-4-isobutyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile;(4Z)-2-[2-(4-chlorophenoxy)pyridin-3-yl]-4-[(dimethylamino)methylene]-1,3-oxazol-5(4H)-one;(5E)-1-(4-methylpentyl)-5-(1H-pyrrol-2-ylmethylene)pyrimidine-2,4,6(1H,3H,5H)-trione;(5E)-3-allyl-5-(2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one;(5E)-5-[4-(diethylamino)benzylidene]-3-{[(2-methoxyphenyl)amino]methyl}-1,3-thiazolidine-2,4-dione;(5R)-5-methyl-4-phenyl-1,3,4-thiadiazolidine-2-thione;(5S,7R)-2,2-dimethyl-5,7-bis(2-phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3-d][1,3]dioxine;(5Z)-5-(4-hydroxybenzylidene)-3-[(2R)-tetrahydrofuran-2-ylmethyl]-2-thioxo-1,3-thiazolidin-4-one;(6E)-5-imino-6-{[1-(2-naphthyl)-1H-pyrrol-2-yl]methylene}-5,6-dihydro-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one;*c1ccccc1C2C(═O)N(C)c3ccccc3C2=O;[(2R)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetic acid;[(5E)-4-oxo-5-(3-thienylmethylene)-2-thioxo-1,3-thiazolidin-3-yl]aceticacid;[(5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]aceticacid;[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-ylethoxy)phenyl]methanone;{4-[(4-methylphenyl)sulfonyl]phenyl}hydrazine;1-(2,4-dihydroxy-6-methylphenyl)-2-phenoxyethanone;1-(2,4-dihydroxyphenyl)-2-(4-isopropylphenoxy)ethanone;1-(3,4-dihydroxyphenyl)-2-({4-[(3,5-dimethoxyphenyl)amino]quinazolin-2-yl}thio)ethanone;1-(4-chlorophenyl)-1-hydroxy-3-phenylurea;1-(4-hydroxy-3,5-dimethylphenyl)-2-[(4-methylphenyl)thio]ethanone;1-(4-iodo-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-ol;1-(5-butyl-2,4-dihydroxyphenyl)-2-pyridin-2-ylethanone;1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)ethanone;1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H-benzimidazol-2-one;1-[4-(7-chloroquinolin-4-yl)piperazino]propan-1-one;1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium;1-ethynyl-2-phenoxybenzene; 1H-perimidine-2-carboxylic acid;2-(1,3-benzodioxol-5-yl)-1-(2,4-dihydroxy-5-propylphenyl)ethanone;2-(1H-benzimidazol-1-yl)-1-(5-ethyl-2,4-dihydroxyphenyl)ethanone;2-(2,4-dichlorophenyl)-1H-imidazo[4,5-b]pyridin-1-ol;2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one;2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one;2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;2-(4-chlorophenyl)-4H-1,3-benzoxazin-4-one;2-(4-chlorophenyl)-5-{[(4-pyridin-3-ylpyrimidin-2-yl)thio]methyl-2,4-dihydro-}H-pyrazol-3-one;2-(4-fluoro-3-phenoxyphenyl)-3-hydroxy-4H-chromen-4-one;2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-N-(4-methylphenyl)acetamide;2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;2-(4-methoxyphenyl)pyridin-3-ol;2-(4-methylphenyl)-4H-1,3-benzoxazin-4-one;2-(morpholin-4-ylmethyl)-1-naphthol;2,2′-buta-1,3-diyne-1,4-diyldiphenol; 2,2′-thiobis(4-chlorophenol);2,4-dichloro-1-naphthyl[2,2,2-trifluoro-1-methyl-1-(trifluoromethyl)ethyl]carbamate;2-[(2-phenoxyethyl)thio]quinazoline-4-thiol;2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-ethylacetamide;2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]-N-1-naphthylacetamide;2-[(5S)-1-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl]phenol;2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium;2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dimethylquinolinium;2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol;2-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol;2-[5-(ethylsulfonyl)-2-hydroxyphenyl]-1H-benzo[de]isoquinoline-1,3(2H)-dione;2-{(1R)-1-[(1-allyl-1H-benzimidazol-2-yl)amino]ethyl}-4-chlorophenol;2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2H)-dione;2-{[5-chloro-6-methyl-2-(2-pyridinyl)-4-pyrimidinyl]sulfanyl}-1-phenyl-1-ethanone;2-amino-1-(2,4-dimethylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carbonitrile;2-amino-5-butyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenylnicotinonitrile;2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one;2-anilino-2-oxoethyl 2-(4-chlorobenzoyl)benzoate;2-chloro-5-phenyl-3-pyridin-4-yl-4H-1,4-thiazine;2-chloro-8-hydroxy-10,10-dimethyl-7-phenylpyrido[1,2-a]indol-6(10H)-one;2-hydrazino-4,6-diphenylpyrimidine; 2-hydrazino-4-methylquinoline;2-hydroxy-N-(4-methylphenyl)benzamide;2-hydroxy-N-(4-propylbenzoyl)benzamide;2-hydroxy-N-[(4-methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]benzamide;2-hydroxy-N-[2-(2-methoxyphenyl)acetyl]benzamide;2-hydroxy-N-{[2-(4-methylphenoxy)-3-pyridinyl]carbonyl}benzamide;2-hydroxy-N-pyridin-3-ylbenzamide; 2-phenyl-4H-thiochromene-4-thione;2-phenyl-5-({[5-(trifluoromethyl)pyridin-2-yl]sulfonyl}methyl)-2,4-dihydro-3H-pyrazol-3-one;2-phenyl-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one;3-(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-N-(2-hydroxyphenyl)-N-(4-methoxybenzyl)propanamide;3-(1-acetyl-1H-indol-3-yl)-4-hydroxy-2H-chromen-2-one;3-(1H-benzimidazol-1-yl)-6-ethyl-7-hydroxy-4H-chromen-4-one;3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-(3-{[(4-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-(4-bromophenyl)-7-hydroxy-2-methyl-4H-chromen-4-one;3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one;3-(5-{(Z)-[5-(4-methylphenyl)-2-oxofuran-3(2H)-ylidene]methyl}-2-furyl)benzoicacid; 3-(quinazolin-4-ylamino)phenyl thiophene-2-carboxylate;3,4-dimethoxy-N-(4-methyl-1,3-benzothiazol-2-yl)benzamide;3,5-dichloro-2-hydroxybenzaldehydeN-tert-butyl-N′-methylthiosemicarbazone;3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phenol;3-[2-(4-methoxyphenyl)ethyl]-10-methyl-6-phenyl-3,4-dihydro-2H,8H-chromeno[6,7-e][1,3]oxazin-8-one;3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propanamide;3-{2-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-1H-pyrrol-1-yl}benzoicacid; 3-benzyl-4-hydroxy-1,2-dihydroquinolin-2-one;3-benzyl-4-hydroxy-1-phenylquinolin-2(1H)-one;3-benzyl-5,6-bis(4-methoxyphenyl)furo[2,3-d]pyrimidin-4(3H)-imine;3-benzyl-5-ethyl-4-hydroxy-6-phenyl-1-(1,3-thiazol-2-yl)pyridin-2(1H)-one;3-benzyl-6-ethoxy-4-hydroxyquinolin-2(1H)-one;3-chloro-N-[2-(methylthio)-1,3-benzothiazol-6-yl]benzamide;3-hydroxy-N-(2-methylphenyl)-2-naphthamide;3-methoxy-2-methyl-6-[1′-phenyl-5-(trifluoromethyl)-1H,1′H-4,4′-bipyrazol-3-yl]phenol;3-methoxy-N-(3-[1,3]oxazolo[4,5-b]pyridin-2-ylphenyl)benzamide;3-methyl-1-[3-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol;3-methyl-1-phenyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]pyridin-6-one;3-methyl-4-[(4-methylphenyl)thio]-1-phenyl-1H-pyrazol-5-ylmethoxyacetate;3-oxo-3-[3-({[3-(trifluoromethyl)phenyl]thio}methyl)phenyl]propanenitrile;4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-indole-2-carbonitrile;4-(1H-benzimidazol-2-yl)-N,N-dimnethylaniline;4-(1-propyl-1H-benzimidazol-2-yl)aniline;4-(2,6-dichlorobenzyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol;4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4,4′-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;4,4′-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(oxy)]diphenol;4,4′-methylenebis(3-hydroxy-2-naphthoicacid)-3,3′-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline(1:1); 4,4′-propane-2,2-diylbis(2-chlorophenol);4-[(3S,6S,7R,8aR)-7-{[2-(4-{(3S,3′S,4′R,6′R,8′R,8a′R)-8′-[(allyloxy)carbonyl]-5-iodo-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazin]-6′-yl}phenoxy)ethoxy]carbonyl}-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trimethylbutan-1-aminium;4-[(4-chlorophenyl)thio]-3-methyl-1-phenyl-1H-pyrazol-5-ol;4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]phenol;4-[1-(4-hydroxy-3-methoxybenzyl)-1H-benzimidazol-2-yl]-2-methoxyphenol;4-[1′-phenyl-5-(trifluoromethyl)-1H,1′H-4,4′-bipyrazol-3-yl]benzene-1,3-diol;4-[4-(1,3-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]benzene-1,3-diol;4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzene-1,3-diol;4-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrazol-5-amine;4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol-2-ylidene]cyclohexa-2,5-dien-1-one;4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en-1-yl]amino}benzoic acid;4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;4-{[(2S)-2-ethylpiperidin-1-yl]methyl}-3-hydroxy-1-methyl-6H-benzo[c]chromen-6-one;4-bromo-2-[(E)-(4H-1,2,4-triazol-4-ylimino)methyl]phenol;4-bromo-2-[5-(2-furyl)-1H-pyrazol-3-yl]phenol;4-bromo-6-chloro-2-oxo-1,3-benzoxathiol-5-yl ethyl carbonate;4-ethyl-6-[4-(1-methyl-1H-benzimidazol-2-yl)-1H-pyrazol-3-yl]benzene-1,3-diol;4-fluoro-N-[3-(trifluoromethyl)phenyl]benzamide;4-hydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-2-one;4-hydroxy-3-propylquinolin-2(1H)-one;4-hydroxy-5-phenyl-6H-pyrido[3,2,1-jk]carbazol-6-one;4-hydroxy-8-methyl-3-{(E)-[(3R)-5-oxo-1,3-diphenylpyrazolidin-4-ylidene]methyl}quinolin-2(1H)-one; 4-phenylquinolin-2-amine;5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-bromo-2-hydroxybenzoyl)-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-carboxamide;5-(benzoylamino)-N,N′-bis(2-hydroxyphenyl)isophthalamide;5-(diethylamino)-2-{(E)-[(2-phenylethyl)imino]methyl}phenol;5,7-dihydroxy-4-propyl-2H-chromen-2-one;5-[(4-methylphenyl)thio]quinazoline-2,4-diamine;5-{2-[(3,4-dichlorophenyl)thio]ethyl}-2-methylpyridine;5-benzyl-3-phenyl-5H-pyrazolo[4,3-c]quinolines;5-benzyl-4-hydroxy-6H-pyrido[3,2,1-jk]carbazol-6-one;5-chloro-2-hydroxy-N-phenylbenzamide;5-hydroxy-4-methyl-7-propyl-2H-chromen-2-one;5-methoxy-2-[3-methyl-4-(1,3-thiazol-4-yl)isoxazol-5-yl]phenol;5-methyl-2-[5-(2-thienyl)-1H-pyrazol-3-yl]phenol;6-(4-chlorophenyl)-7-hydroxy-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione″6,6′-biquinoline;6-[(S)-[4-(dimethylamino)phenyl](piperidin-1-yl)methyl]-1,3-benzodioxol-5-ol;6-{[(2-ethylphenyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;6-{[(4-chlorophenyl)thio]methyl}-2-phenyl-1H-pyrazolo[3,4-b]pyridine-3,4(2H,7H)-dione;6-{[(4-ethoxyphenyl)(methyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;6-allyl-7-hydroxy-4,8-dimethyl-2H-chromen-2-one;6-amino-1-ethylbenzo[cd]indol-2(1H)-one;6-benzyl-7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one;6-bromo-2-(trifluoromethyl)quinolin-4-ol;6-butyl-2-(2-furyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-7-one;6-chloro-2-(4-chlorophenyl)-1H-benzimidazol-1-ol;6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide;6-chloro-3-(4-methylphenyl)-3,4-dihydro-2H-1,3-benzoxazine;6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;6-ethyl-7-hydroxy-3-(1-methyl-1H-benzimidazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one;6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one;6-methoxy-N-[(1S)-1-methylpropyl]furo[2,3-b]quinoline-2-carboxamide;6-phenyl[1,2,3,4]tetraazolo[1,5-b]pyridazin-7-ol;7-(4-bromophenyl)-5-hydroxy-1,3-benzoxathiol-2-one;7,8-dihydroxy-2-phenyl-4H-chromen-4-one;7,8-dihydroxy-4-phenyl-2H-chromen-2-one;7-[(2E)-2-(4-fluoro-3-phenoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-[2-chloro-5-(trifluoromethyl)phenyl]-5-hydroxy-1,3-benzoxathiol-2-one;7-{(2E)-2-[(2-fluorobiphenyl-4-yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-benzyl-8-hydroxy-10,10-dimethyl-6,10-dihydropyrido[1,2-a]indol-6-one;7-chloro-4-piperidinoquinoline;7-chloro-N-(3-fluoro-4-methylphenyl)quinolin-4-amine;7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carboxamide;7-chloro-N-phenylquinolin-4-amine;7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;7-hydroxy-5-methyl-3-(1-phenyl-1H-pyrazol-4-yl)-2-(trifluoromethyl)-4H-chromen-4-one;7-hydroxy-6-methyl-3-(4-methyl-1,3-thiazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one;8-(trifluoromethoxy)-2-(trifluoromethyl)quinolin-4-ol;8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;8-[(2E)-2-(5-bromo-2-methoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;8-{(2E)-2-[(6-bromo-1,3-benzodioxol-5-yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-8-oxooctanamide;8-methoxy-N,N-dimethyl-5H-pyrimido[5,4-b]indol-4-amine;allyl(3R,3′R,4′S,6′R,8′S,8a′S)-6′-{4-[2-({[(3S,6R,7S,8aS)-3-[4-(dimethylamino)butyl]-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7-yl]carbonyl}oxy)ethoxy]phenyl}-5-iodo-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazine]-8′-carboxylate″bis[4-(dimethylamino)phenyl]methanoneoxime; CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;COC(═O)[C@]1(Cc2ccc(O)c(CC═C(C)C)c2)OC(═O)C(═C1c3ccc(O)cc3)O;COc1cc(/C═C/2\Oc3cc(O)ccc3C2=O)ccc1O;COc1cc(ccc1O)c2oc3cc(O)cc(O)c3c(═O)c2O;COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;ethyl(2E)-3-(2-hydroxy-5-nitrophenyl)acrylate; ethyl1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxylate;ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate; ethyl4-(benzylamino)-6-ethoxyquinoline-3-carboxylate; ethyl4-[({[(5R)-5-ethyl-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-2-yl]thio}acetyl)amino]benzoate;ethyl 4-[(2-phenylethyl)amino]quinoline-3-carboxylate; ethyl4-{[(2-anilino-2-oxoethyl)thio]methyl}-5-hydroxy-2-phenyl-1-benzofuran-3-carboxylate;ethyl 4-{[(2E)-3-(2-thienyl)prop-2-enoyl]amino}benzoate; ethyl6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-{[(4-methylphenyl)thio]methyl}-1H-indole-3-carboxylate;ethyl 6-ethoxy-4-{[(1R)-1-methylpropyl]amino}quinoline-3-carboxylate;ethyl 6-methyl-4-[(4-morpholin-4-ylphenyl)amino]quinoline-3-carboxylate;isopropyl(2S)-2-{[(2S)-2-{[(2S,3R)-2-{[(2S)-2-amino-3-mercaptopropyl]amino}-3-methylpentyl]oxy}-3-phenylpropanoyl]amino}-4-(methylsulfonyl)butanoate;methyl(2Z)-2-(4-hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5-carboxylate;methyl 1-hydroxy-3-methylpyrido[1,2-a]benzimidazole-4-carboxylate;methyl2,3-bis-O-(biphenyl-2-ylcarbamoyl)-4-O-[(3-ethylphenyl)carbamoyl]-alpha-L-idopyranoside;methyl5-hydroxy-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate;N-(1,3-benzodioxol-5-yl)-7-chloroquinolin-4-amine;N-(2,3-dihydro-1-benzofuran-5-ylcarbonyl)-2-hydroxybenzamide;N-(2,5-dimethylphenyl)benzamide;N-(2-chlorobenzyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;N-(2-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-sulfonamide;N-(2-ethoxyphenyl)-2-hydroxybenzamide;N-(2-hydroxy-4-methylphenyl)-4-[(methylthio)methyl]benzamide;N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;N-(2-hydroxybenzoyl)-3-methoxybenzamide;N-(2-hydroxybenzoyl)-4-(trifluoromethyl)benzamide;N-(2-hydroxyphenyl)-8-[(2E)-2-(1-naphthylmethylene)hydrazino]-8-oxooctanamide;N-(3-bromo-4-hydroxy-1-naphthyl)-4-chlorobenzenesulfonamide;N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;N-(3-chlorophenyl)-4-(5-hydroxy-1-phenyl-1H-pyrazol-3-yl)piperidine-1-carbothioamide;N-(3-furylcarbonyl)-2-hydroxybenzamide;N-(3-hydroxypyridin-2-yl)-4-phenoxybenzamide;N-(3-imidazo[1,2-a]pyrimidin-2-ylphenyl)cyclopentanecarboxamide;N-(4-bromophenyl)-2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]acetamide;N-(4-carbamoylphenyl)-1-phenyl-3-(2-thienyl)-1H-pyrazole-4-carboxamide;N-(4-ethylbenzoyl)-2-hydroxybenzamide;N-(4-fluorophenyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;N-(5-{[(1S)-1-methylpropyl]thio}-1,3,4-thiadiazol-2-yl)-2-(trifluoromethyl)benzamide;N-(5-hydroxy-1-naphthyl)-4-methylbenzenesulfonamide;N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;N-(6-methyl-1,3-benzothiazol-2(3H)-ylidene)thiophene-2-carboxamide;N-(cyclohexylcarbonyl)-2-hydroxybenzamide;N,2-diphenylquinazolin-4-amine;N,N,8-trimethyl-5H-pyrimido[5,4-b]indol-4-amine;N,N′-1H-isoindole-1,3(2H)-diylidenedianiline;N,N-diethyl-8-methyl-5H-pyrimido[5,4-b]indol-4-amine;N,N-dimethyl-4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzenesulfonamide;N-[(1E)-(9-ethyl-9H-carbazol-3-yl)methylene]-4H-1,2,4-triazol-4-amine;N-[(1E)-1H-indol-3-ylmethylene]-1-propyl-1H-benzimidazol-2-amine;N-[(1S)-1-benzylpropyl]-6-[(4-methylpiperidin-1-yl)sulfonyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide;N-[(1S)-1-phenylethyl]quinazolin-4-amine;N-[(3-chloro-1-benzothiophen-2-yl)carbonyl]-2-hydroxybenzamide;N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethanamine;N-[2-(1H-benzimidazol-2-yl)phenyl]-2-methylpropanamide;N-[2-chloro-5-(trifluoromethyl)phenyl]-2-(4,4-dimethyl-2,6-dioxocyclohexyl)acetamide;N-[2-hydroxy-3-(4-oxo-4H-chromen-2-yl)phenyl]acetamide;N-[3-(1,3-benzothiazol-2-yl)-4-hydroxyphenyl]-2,2-dimethylpropanamide;N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]benzenesulfonamide;N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]thiophene-2-sulfonamide;N-[4-(1H-benzimidazol-2-yl)phenyl]-2-(2-methoxyphenyl)acetamide;N-[4-(ethylsulfonyl)-2-hydroxyphenyl]benzamide;N-[5-(ethylsulfonyl)-2-hydroxyphenyl]-2-(4-methoxyphenoxy)acetamide;N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;N-benzo[g]quinolin-4-yl-N′-isopropylbenzene-1,4-diamine;N-benzyl-2-hydroxybenzamide;N-benzyl-6-{[(3-methoxyphenyl)amino]sulfonyl}-N-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide;N-ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)/C═C/c4ccccc4)c(O)c3)[C@@H]1O;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O)cc(O)c4C3=O)c5ccc(O)cc5)c(O)cc(O)c2C1=O)c6ccc(O)cc6;O[C@H]1[C@H]2[C@H](CC(═O)O)C(═O)O[C@@H]3C(COC(═O)c4cc(O)c(O)c(O)c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O)c6cc(O)c(O)c(OC1=O)c26)[C@@H]3OC(═O)c7cc(O)c(O)c(O)c7;OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O)cc(O)c4c3=O)c5ccc(O)cc5)[C@H](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O;andOC1[C@H](OC(═O)c2cc(O)c(O)c(O)c2)OC3COC(═O)c4cc(O)c(O)c(O)c4-c5c(O)c(O)c(O)cc5C(═O)O[C@@H]1[C@@H]3OC(═O)c6cc(O)c(O)c(O)c6c7c(O)c(O)c(O)cc7C(═O)OOc1ccc(cc1)[C@H]2CC(═O)c3c(O)cc(O)c([C@H]4[C@@H](Oc5cc(O)cc(O)c5C4=O)c6ccc(O)cc6)c3020c1ccc2C(═O)/C(═C/c3ccc(O)c(O)c3)/Oc2c1.
 38. A method foridentifying compounds that inhibit HDP expression, comprising the stepsof a) contacting Plasmodium with a test compound and b) determiningwhether said Plasmodium expresses HDP.
 39. The method of claim 38,wherein said step of determining is carried out by measuring mRNA. 40.The method of claim 38, wherein said step of determining is carried outby measuring HDP.
 41. A pharmaceutical composition comprising apharmaceutically acceptable carrier and an antimalarially effectiveamount of at least one compound selected from the group consisting of:(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one;(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide;(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-6-ol;(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one;[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-ylethoxy)phenyl]methanone;1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H-benzimidazol-2-one;1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium;1H-perimidine-2-carboxylic acid;2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;2-(4-methoxyphenyl)pyridin-3-ol; 2-(morpholin-4-ylmethyl)-1-naphthol;2,2′-buta-1,3-diyne-1,4-diyldiphenol;2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium;2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dimethylquinolinium;2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;2-[4-(1-benzofuran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol;2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl-1H-isoindole-1,3(2H)-dione;2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one;2-hydrazino-4-methylquinoline; 2-hydroxy-N-(4-propylbenzoyl)benzamide;3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phenol;3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propanamide;4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyloxy)-1H-indole-2-carbonitrile;4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4,4′-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;4,4′-methylenebis(3-hydroxy-2-naphthoicacid)-3,3′-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline(1:1); 4,4′-propane-2,2-diylbis(2-chlorophenol);4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]phenol;4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzene-1,3-diol;4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol-2-ylidene]cyclohexa-2,5-dien-1-one;4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;4-phenylquinolin-2-amine;5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-carboxamide;5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-4H-chromen-4-one;6-amino-1-ethylbenzo[cd]indol-2(1H)-one;6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-chloro-N-[2-(dimethylarnino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carboxamide;7-chloro-N-phenylquinolin-4-amine;7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1; COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;ethyl1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxylate;ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate;N-(2-ethoxyphenyl)-2-hydroxybenzarnide;N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;N-(2-hydroxybenzoyl)-3-methoxybenzamide;N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;N-(3-furylcarbonyl)-2-hydroxybenzamide;N-(4-ethylbenzoyl)-2-hydroxybenzamide;N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;N-[(1S)-1-phenylethyl]quinazolin-4-amine;N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethanamine;N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diarnine;N-benzo[g]quinolin-4-yl-N′-isopropylbenzene-1,4-diamine;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(o)c(O)c2)O[C@@H](Oc3ccc(C(═O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;and O[C@H]1[C@H]2[C@H](CC(═O)O)C(═O)O[C@@H]3C(COC(═O)c4cc(O)c(O)c(O)c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O)c6cc(O)c(O)c(OC1=O)c26)[C(@H]3OC(═O)c7cc(O)c(O)c(O)c7.42. A pharmaceutical composition comprising a pharmnaceuticallyacceptable carrier and an antimalarially effective amount of at leastone compound selected from the group consisting of(10S)-10-(dimethylamino)-9-methyl-7H,10H-naphtho[1,8-gh]chromen-7-one;(1E,4E)-1-[4-(dimethylamino)phenyl]-5-(3,4,5-trimethoxyphenyl)penta-1,4-dien-3-one;(1R,3R)-1-isopropyl-2,3,4,9-tetrahydro-1H-beta-carboline-3-carboxylicacid;(1S)-1-(3,4-dichlorophenyl)-2-(2-imino-1,3-benzothiazol-3(2H)-yl)ethanol;(2E)-2-[(pyridin-3-ylamino)methylene]-1-benzothiophen-3(2H)-one;(2E)-3-(3,4-dimethoxyphenyl)-N-(3,4-dimethylphenyl)acrylamide;(2E)-6-ethoxy-2-(2-hydroxybenzylidene)-1-benzothiophen-3(2H)-one;(2E)-N-(2-methyl-1,3-benzothiazol-6-yl)-3-phenylacrylamide;(2E,5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-2-(phenylimino)-1,3-thiazolidin-4-one;(2E,5Z)-2-[(2-chlorophenyl)imino]-5-(4-hydroxy-3-nitrobenzylidene)-1,3-thiazolidin-4-one;(2R)-1-(benzylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol;(2R)-2-(2,4-dichlorophenoxy)-N-(5-oxo-1-phenyl-4,5-dihydro-1H-pyrazol-3-yl)propanamide;(2R)-2-[(5Z)-5-(4-hydroxy-3,5-dimethoxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]-3-methylbutanoicacid;(2R)-2-[(E)-2-(1,3-benzodioxol-5-yl)vinyl]-5,6-dimethyl-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one;(2R,3Z)-6-chloro-3-[(dimethylarnino)methylene]-2-methyl-2,3-dihydro-4H-thiochromen-4-one;(2S)-2-(4-chlorophenyl)-3-oxo-4-phenylbutanenitrile;(2S)-2-[(5E)-5-(1H-indol-3-ylmethylene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]succinicacid;(2S)-N-(4-chlorophenyl)-2-methyl-2,3-dihydro-4H-1,4-benzoxazine-4-carboxamide;(2S,3Z)-3-(1H-indol-3-ylmethylene)-2-phenyl-2,3-dihydro-4H-chromen-4-one;(2Z)-2-acetamido-N-(3,5-dimethylphenyl)-3-phenylacrylamide;(2Z,5E)-2-[(3,5-dimethylphenyl)imino]-5-(2-hydroxy-3-methoxybenzylidene)-1,3-thiazolidin-4-one;(2Z,5Z)-2-[(2-chlorophenyl)imino]-5-(2-hydroxy-3-nitrobenzylidene)-1,3-thiazolidin-4-one;(3,5-dichloro-2-hydroxyphenyl)(isoxazol-4-yl)methanone;(3-{(E)-[1-(3-fluorophenyl)-2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene]methyl}-1H-indol-1-yl)acetonitrile;(3aR,4R,9bR)-8-chloro-4-(4-chlorophenyl)-9-nitro-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinolin-6-ol;(3aS,6aS)-3-benzoyl-1,5-diphenyl-3a,6a-dihydropyrrolo[3,4-c]pyrazole-4,6(1H,5H)-dione;(3R)-3-(2-hydroxy-4-methylphenyl)-N-(2-methoxyphenyl)-3-phenylpropanamide;(3R)-5,7-dichloro-3-hydroxy-3-(2-methylimidazo[1,2-a]pyridin-3-yl)-1,3-dihydro-2H-indol-2-one;(3R,3′R,4′S,6′R,8′S,8a′S)-5-(4-hydroxybut-1-yn-1-yl)-6′-[4-(2-hydroxyethoxy)phenyl]-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazine]-8′-carboxylic acid;(3S)-3-(2-hydroxy-4-methylbenzoyl)-2-(4-methylphenyl)isoindolin-1-one;(3S,3aR,6aR)-3-(5-bromo-2-hydroxyphenyl)-5-butyl-2-phenyldihydro-2H-pyrrolo[3,4-d]isoxazole-4,6(3H,5H)-dione;(3S,6S,7R,8aR)-3-(4-acetamidobutyl)-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazine-7-carboxylicacid;(3Z)-3-(3-hydroxy-4-methoxybenzylidene)-1-methyl-1,3-dihydro-2H-indol-2-one;(4E)-2-(4-methoxyphenyl)-4-[(4-methoxyphenyl)imino]-4H-chromen-6-ol;(4R)-3-(3,4-dichlorophenyl)-4-hydroxy-N-isopropyl-2-oxo-1,2,3,4-tetrahydroquinazoline-4-carboxamide;(4R)-4-(4-bromophenyl)-3-hydroxy-1-isopropyl-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7(6H)-one;(4R)-4-(4-ethylphenyl)-3-hydroxy-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one;(4R)-5-(2-furylmethyl)-3-(2-hydroxyphenyl)-4-phenyl-4,5-dihydropyrrolo[3,4-c]pyrazol-6(1H)-one;(4R)-N˜4˜-(6-chloro-2-methoxyacridin-9-yl)-N˜1˜,N˜1˜-diethylpentane-1,4-diamine;(4S)-4-(2-bromobenzoyl)-5-methyl-2-phenyl-2,4-dihydro-3H-pyrazol-3-one;(4S)-4-(2-furyl)-3-hydroxy-7,7-dimethyl-2-phenyl-2,4,6,7,8,9-hexahydro-5H-pyrazolo[3,4-b]quinolin-5-one;(4S)-5,7-dihydroxy-4-phenylchroman-2-one;(4S,5S)-3,5-diphenyl-4,5-dihydro-1H-pyrazol-4-ol;(4S,7R)-2-amino-4-isobutyl-5-oxo-7-phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile;(4Z)-2-[2-(4-chlorophenoxy)pyridin-3-yl]-4-[(dimethylamino)methylene]-1,3-oxazol-5(4H)-one;(5E)-1-(4-methylpentyl)-5-(1H-pyrrol-2-ylmethylene)pyrimidine-2,4,6(1H,3H,5H)-trione;(5E)-3-allyl-5-(2-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one;(5E)-5-[4-(diethylamino)benzylidene]-3-{[(2-methoxyphenyl)amino]methyl}-1,3-thiazolidine-2,4-dione;(5R)-5-methyl-4-phenyl-1,3,4-thiadiazolidine-2-thione;(5S,7R)-2,2-dimethyl-5,7-bis(2-phenylethyl)-7,8-dihydro-4H,5H-pyrano[4,3-d][1,3]dioxine;(5Z)-5-(4-hydroxybenzylidene)-3-[(2R)-tetrahydrofuran-2-ylmethyl]-2-thioxo-1,3-thiazolidin-4-one;(6E)-5-imino-6-{[1-(2-naphthyl)-1H-pyrrol-2-yl]methylene}-5,6-dihydro-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidin-7-one;*c1ccccc1C2C(═O)N(C)c3ccccc3C2=O;[(2R)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetic acid;[(5E)-4-oxo-5-(3-thienylmethylene)-2-thioxo-1,3-thiazolidin-3-yl]aceticacid;[(5E)-5-(5-bromo-3-chloro-2-hydroxybenzylidene)-4-oxo-2-thioxo-1,3-thiazolidin-3-yl]aceticacid;[6-hydroxy-2-(4-hydroxyphenyl)-1-benzothien-3-yl][4-(2-piperidin-1-ylethoxy)phenyl]methanone;{4-[(4-methylphenyl)sulfonyl]phenyl}hydrazine;1-(2,4-dihydroxy-6-methylphenyl)-2-phenoxyethanone;1-(2,4-dihydroxyphenyl)-2-(4-isopropylphenoxy)ethanone;1-(3,4-dihydroxyphenyl)-2-({4-[(3,5-dimethoxyphenyl)amino]quinazolin-2-yl}thio)ethanone;1-(4-chlorophenyl)-1-hydroxy-3-phenylurea;1-(4-hydroxy-3,5-dimethylphenyl)-2-[(4-methylphenyl)thio]ethanone;1-(4-iodo-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-ol;1-(5-butyl-2,4-dihydroxyphenyl)-2-pyridin-2-ylethanone;1-(5-ethyl-2,4-dihydroxyphenyl)-2-(1-methyl-1H-benzimidazol-2-yl)ethanone;1,2,3,4,6-pentakis-O-(3,4,5-trihydroxybenzoyl)-beta-D-glucopyranose;1-[(2S)-3-(9H-carbazol-9-yl)-2-hydroxypropyl]-8-hydroxyquinolinium;1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-1,3-dihydro-2H-benzimidazol-2-one;1-[4-(7-chloroquinolin-4-yl)piperazino]propan-1-one;1-ethyl-6-methoxy-4-methyl-2-[(Z)-(3-methyl-1,3-thiazol-2(3H)-ylidene)methyl]benzo[h]quinolinium;1-ethynyl-2-phenoxybenzene; 1H-perimidine-2-carboxylic acid;2-(1,3-benzodioxol-5-yl)-1-(2,4-dihydroxy-5-propylphenyl)ethanone;2-(1H-benzimidazol-1-yl)-1-(5-ethyl-2,4-dihydroxyphenyl)ethanone;2-(2,4-dichlorophenyl)-1H-imidazo[4,5-b]pyridin-1-ol;2-(2,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one;2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen-4-one;2-(3,4-dihydroxyphenyl)-3,7-dihydroxy-4H-chromen-4-one;2-(4-chlorophenyl)-4H-1,3-benzoxazin-4-one;2-(4-chlorophenyl)-5-{[(4-pyridin-3-ylpyrimidin-2-yl)thio]methyl}-2,4-dihydro-3H-pyrazol-3-one;2-(4-fluoro-3-phenoxyphenyl)-3-hydroxy-4H-chromen-4-one;2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)-N-(4-methylphenyl)acetamide;2-(4-methoxyphenyl)-4H-1,3-benzoxazin-4-one;2-(4-methoxyphenyl)pyridin-3-ol;2-(4-methylphenyl)-4H-1,3-benzoxazin-4-one;2-(morpholin-4-ylmethyl)-1-naphthol;2,2′-buta-1,3-diyne-1,4-diyldiphenol; 2,2′-thiobis(4-chlorophenol);2,4-dichloro-1-naphthyl[2,2,2-trifluoro-1-methyl-1-(trifluoromethyl)ethyl]carbamate;2-[(2-phenoxyethyl)thio]quinazoline-4-thiol;2-[(2R)-6,7-dimethyl-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]-N-ethylacetamide;2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]-N-1-naphthylacetamide;2-[(5S)-1-(4-nitrophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-5-yl]phenol;2-[(E)-(6-methoxy-1-methylquinolin-2(1H)-ylidene)methyl]-3-methyl-1,3-benzothiazol-3-ium;2-[(Z)-(3-ethyl-6-methoxy-1,3-benzothiazol-2(3H)-ylidene)methyl]-1,6-dimethylquinolinium;2-[2-(4-hydroxyphenyl)ethyl]-6-methylpyridin-3-ol;2-[4-(1-benzofuiran-2-yl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-5-methoxyphenol;2-[5-(2-methoxyphenyl)-1,3,4-oxadiazol-2-yl]phenol;2-[5-(ethylsulfonyl)-2-hydroxyphenyl]-1H-benzo[de]isoquinoline-1,3(2H)-dione;2-{(1R)-1-[(1-allyl-1H-benzimidazol-2-yl)amino]ethyl}-4-chlorophenol;2-{(3R)-1-[4-(2-hydroxyethoxy)benzyl]piperidin-3-yl}-1H-isoindole-1,3(2H)-dione;2-{[5-chloro-6-methyl-2-(2-pyridinyl)-4-pyrimidinyl]sulfanyl}-1-phenyl-1-ethanone;2-amino-1-(2,4-dimethylphenyl)-1H-pyrrolo[2,3-b]quinoxaline-3-carbonitrile;2-amino-5-butyl-4-(4-hydroxy-3-methoxyphenyl)-6-phenylnicotinonitrile;2-amino-8-(azepan-1-ylmethyl)-3-(1,3-benzothiazol-2-yl)-7-hydroxy-4H-chromen-4-one;2-anilino-2-oxoethyl 2-(4-chlorobenzoyl)benzoate;2-chloro-5-phenyl-3-pyridin-4-yl-4H-1,4-thiazine;2-chloro-8-hydroxy-10,10-dimethyl-7-phenylpyrido[1,2-a]indol-6(10H)-one;2-hydrazino-4,6-diphenylpyrimidine; 2-hydrazino-4-methylquinoline;2-hydroxy-N-(4-methylphenyl)benzamide;2-hydroxy-N-(4-propylbenzoyl)benzamide;2-hydroxy-N-[(4-methyl-2-phenyl-1,3-thiazol-5-yl)carbonyl]benzamide;2-hydroxy-N-[2-(2-methoxyphenyl)acetyl]benzamide;2-hydroxy-N-{[2-(4-methylphenoxy)-3-pyridinyl]carbonyl}benzamide;2-hydroxy-N-pyridin-3-ylbenzamide; 2-phenyl-4H-thiochromene-4-thione;2-phenyl-5-({[5-(trifluoromethyl)pyridin-2-yl]sulfonyl}methyl)-2,4-dihydro-3H-pyrazol-3-one;2-phenyl-5-(trifluoromethyl)-2,4-dihydro-3H-pyrazol-3-one;3-(1,1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)-N-(2-hydroxyphenyl)-N-(4-methoxybenzyl)propanamide;3-(1-acetyl-1H-indol-3-yl)-4-hydroxy-2H-chromen-2-one;3-(1H-benzimidazol-1-yl)-6-ethyl-7-hydroxy-4H-chromen-4-one;3-(2-hydroxy-5-methoxybenzoyl)-2-(4-methylphenyl)isoindolin-1-one;3-(3-{[(2-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-(3-{[(4-chlorophenyl)thio]methyl}phenyl)-3-oxopropanenitrile;3-(4-bromophenyl)-7-hydroxy-2-methyl-4H-chromen-4-one;3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)propan-1-one;3-(5-{(Z)-[5-(4-methylphenyl)-2-oxofuran-3(2H)-ylidene]methyl}-2-furyl)benzoicacid; 3-(quinazolin-4-ylamino)phenyl thiophene-2-carboxylate;3,4-dimethoxy-N-(4-methyl-1,3-benzothiazol-2-yl)benzamide;3,5-dichloro-2-hydroxybenzaldehydeN-tert-butyl-N′-methylthiosemicarbazone;3-[(4-{[(2R)-tetrahydrofuran-2-ylmethyl]amino}quinazolin-2-yl)amino]phenol;3-[2-(4-methoxyphenyl)ethyl]-10-methyl-6-phenyl-3,4-dihydro-2H,8H-chromeno[6,7-e][1,3]oxazin-8-one;3-[4-(9H-fluoren-9-yl)piperazin-1-yl]-N-[3-(trifluoromethyl)phenyl]propanamide;3-{2-[(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)methyl]-1H-pyrrol-1-yl}benzoicacid; 3-benzyl-4-hydroxy-1,2-dihydroquinolin-2-one;3-benzyl-4-hydroxy-1-phenylquinolin-2(1H)-one;3-benzyl-5,6-bis(4-methoxyphenyl)furo[2,3-d]pyrimidin-4(3H)-imine;3-benzyl-5-ethyl-4-hydroxy-6-phenyl-1-(1,3-thiazol-2-yl)pyridin-2(1H)-one;3-benzyl-6-ethoxy-4-hydroxyquinolin-2(1H)-one;3-chloro-N-[2-(methylthio)-1,3-benzothiazol-6-yl]benzamide;3-hydroxy-N-(2-methylphenyl)-2-naphthamide;3-methoxy-2-methyl-6-[1′-phenyl-5-(trifluoromethyl)-1H,1′H-4,4′-bipyrazol-3-yl]phenol;3-methoxy-N-(3-[1,3]oxazolo[4,5-b]pyridin-2-ylphenyl)benzamide;3-methyl-1-[3-(trifluoromethyl)phenyl]-1H-pyrazol-5-ol;3-methyl-1-phenyl-4-(trifluoromethyl)-1,7-dihydro-6H-pyrazolo[3,4-b]pyridin-6-one;3-methyl-4-[(4-methylphenyl)thio]-1-phenyl-1H-pyrazol-5-ylmethoxyacetate;3-oxo-3-[3-({[3-(trifluoromethyl)phenyl]thio}methyl)phenyl]propanenitrile;4-({(2S)-3-[4-(diphenylmethyl)piperazin-1-yl]-2-hydroxypropyl}oxy)-1H-indole-2-carbonitrile;4-(1H-benzimidazol-2-yl)-N,N-dimethylaniline;4-(1-propyl-1H-benzimidazol-2-yl)aniline;4-(2,6-dichlorobenzyl)-3-methyl-1-phenyl-1H-pyrazol-5-ol;4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4-(7-methylimidazo[1,2-a]pyridin-2-yl)benzene-1,2-diol;4,4′-(4-phenyl-1H-imidazole-2,5-diyl)diphenol;4,4′-[(2,3,5,6-tetrafluoro-1,4-phenylene)bis(oxy)]diphenol;4,4′-methylenebis(3-hydroxy-2-naphthoicacid)-3,3′-[(4-iminocyclohexa-2,5-dien-1-ylidene)methylene]dianiline(1:1); 4,4′-propane-2,2-diylbis(2-chlorophenol);4-[(3S,6S,7R,8aR)-7-{[2-(4-{(3S,3′S,4′R,6′R,8′R,8a′R)-8′-[(allyloxy)carbonyl]-5-iodo-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazin]-6′-yl}phenoxy)ethoxy]carbonyl}-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-3-yl]-N,N,N-trimethylbutan-1-aminium;4-[(4-chlorophenyl)thio]-3-methyl-1-phenyl-1H-pyrazol-5-ol;4-[(5S)-5-(4-fluorophenyl)-1-(4-nitrophenyl)-4,5-dihydro-1H-pyrazol-3-yl]phenol;4-[1-(4-hydroxy-3-methoxybenzyl)-1H-benzimidazol-2-yl]-2-methoxyphenol;4-[1′-phenyl-5-(trifluoromethyl)-1H,1′H-4,4′-bipyrazol-3-yl]benzene-1,3-diol;4-[4-(1,3-benzothiazol-2-yl)-5-methyl-1H-pyrazol-3-yl]benzene-1,3-diol;4-[4-(3,4-dihydro-2H-1,5-benzodioxepin-7-yl)-3-methylisoxazol-5-yl]benzene-1,3-diol;4-[4-(4-chlorophenyl)-1,3-thiazol-2-yl]-1-methyl-1H-pyrazol-5-amine;4-[5-[5-(4-methylpiperazin-1-yl)-3H-benzoimidazol-2-yl]-1,3-dihydrobenzoimidazol-2-ylidene]cyclohexa-2,5-dien-1-one;4-{[(1E)-3-(2-furyl)-3-oxoprop-1-en-1-yl]amino}benzoic acid;4-{[(2-ethylphenyl)amino]methyl}-5-(hydroxymethyl)-2-methylpyridin-3-ol;4-{[(2S)-2-ethylpiperidin-1-yl]methyl}-3-hydroxy-1-methyl-6H-benzo[c]chromen-6-one;4-bromo-2-[(E)-(4H-1,2,4-triazol-4-ylimino)methyl]phenol;4-bromo-2-[5-(2-furyl)-1H-pyrazol-3-yl]phenol;4-bromo-6-chloro-2-oxo-1,3-benzoxathiol-5-yl ethyl carbonate;4-ethyl-6-[4-(1-methyl-H 1H-benzimidazol-2-yl)-H1H-pyrazol-3-yl]benzene-1,3-diol;4-fluoro-N-[3-(trifluoromethyl)phenyl]benzamide;4-hydroxy-3-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl]-2H-chromen-2-one;4-hydroxy-3-propylquinolin-2(11)-one;4-hydroxy-5-phenyl-6H-pyrido[3,2,1-jk]carbazol-6-one;4-hydroxy-8-methyl-3-{(E)-[(3R)-5-oxo-1,3-diphenylpyrazolidin-4-ylidene]methyl}quinolin-2(1H)-one;4-phenylquinolin-2-amine;5-(2-hydroxy-5-methylbenzoyl)-1-(4-methylphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-bromo-2-hydroxybenzoyl)-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile;5-(5-chloro-2-hydroxybenzoyl)-2-oxo-N,1-diphenyl-1,2-dihydropyridine-3-carboxamide;5-(benzoylamino)-N,N′-bis(2-hydroxyphenyl)isophthalamide;5-(diethylamino)-2-{(E)-[(2-phenylethyl)imino]methyl}phenol;5,7-dihydroxy-4-propyl-2H-chromen-2-one;5-[(4-methylphenyl)thio]quinazoline-2,4-diamine;5-{2-[(3,4-dichlorophenyl)thio]ethyl}-2-methylpyridine;5-benzyl-3-phenyl-5H-pyrazolo[4,3-c]quinolines;5-benzyl-4-hydroxy-6H-pyrido[3,2,1-jk]carbazol-6-one;5-chloro-2-hydroxy-N-phenylbenzamide;5-hydroxy-4-methyl-7-propyl-2H-chromen-2-one;5-methoxy-2-[3-methyl-4-(1,3-thiazol-4-yl)isoxazol-5-yl]phenol;5-methyl-2-[5-(2-thienyl)-1H-pyrazol-3-yl]phenol;6-(4-chlorophenyl)-7-hydroxy-1,3-dimethyl-1H-pyrrolo[3,2-d]pyrimidine-2,4(3H,5H)-dione″6,6′-biquinoline;6-[(S)-[4-(dimethylamino)phenyl](piperidin-1-yl)methyl]-1,3-benzodioxol-5-ol;6-{[(2-ethylphenyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;6-{[(4-chlorophenyl)thio]methyl}-2-phenyl-1H-pyrazolo[3,4-b]pyridine-3,4(2H,7H)-dione;6-{[(4-ethoxyphenyl)(methyl)amino]sulfonyl}-4-oxo-N-[(2S)-tetrahydrofuran-2-ylmethyl]-1,4-dihydroquinoline-3-carboxamide;6-allyl-7-hydroxy-4, 8-dimethyl-2H-chromen-2-one;6-amino-1-ethylbenzo[cd]indol-2(1H)-one;6-benzyl-7-hydroxy-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-5-one;6-bromo-2-(trifluoromethyl)quinolin-4-ol;6-butyl-2-(2-furyl)-5-methyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-7-one;6-chloro-2-(4-chlorophenyl)-1H-benzimidazol-1-ol;6-chloro-2H-1,2,4-benzothiadiazine-7-sulfonamide 1,1-dioxide;6-chloro-3-(4-methylphenyl)-3,4-dihydro-2H-1,3-benzoxazine;6-chloro-3-[2-(4-chlorophenyl)ethyl]-3,4-dihydro-2H-1,3-benzoxazine;6-ethyl-7-hydroxy-3-(1-methyl-1H-benzimidazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one;6-fluoro-4-hydroxy-3-phenylquinolin-2(1H)-one;6-methoxy-N-[(1S)-1-methylpropyl]furo[2,3-b]quinoline-2-carboxamide;6-phenyl[1,2,3,4]tetraazolo[1,5-b]pyridazin-7-ol;7-(4-bromophenyl)-5-hydroxy-1,3-benzoxathiol-2-one;7,8-dihydroxy-2-phenyl-4H-chromen-4-one;7,8-dihydroxy-4-phenyl-2H-chromen-2-one;7-[(2E)-2-(4-fluoro-3-phenoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-[(2E)-2-(biphenyl-4-ylmethylene)hydrazino]-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-[2-chloro-5-(trifluoromethyl)phenyl]-5-hydroxy-1,3-benzoxathiol-2-one;7-{(2E)-2-[(2-fluorobiphenyl-4-yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-7-oxoheptanamide;7-benzyl-8-hydroxy-10,10-dimethyl-6,10-dihydropyrido[1,2-a]indol-6-one;7-chloro-4-piperidinoquinoline;7-chloro-N-(3-fluoro-4-methylphenyl)quinolin-4-amine;7-chloro-N-[2-(dimethylamino)ethyl]-4H-thieno[3,2-c]thiochromene-2-carboxamide;7-chloro-N-phenylquinolin-4-amine;7-hydroxy-2-methyl-6-propyl-3-pyridin-2-yl-4H-chromen-4-one;7-hydroxy-5-methy1-3-(1-phenyl-1H-pyrazol-4-yl)-2-(trifluoromethyl)-4H-chromen-4-one;7-hydroxy-6-methyl-3-(4-methyl-1,3-thiazol-2-yl)-2-(trifluoromethyl)-4H-chromen-4-one;8-(trifluoromethoxy)-2-(trifluoromethyl)quinolin-4-ol;8-[(2E)-2-(2-bromobenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;8-[(2E)-2-(5-bromo-2-methoxybenzylidene)hydrazino]-N-(2-hydroxyphenyl)-8-oxooctanamide;8-{(2E)-2-[(6-bromo-1,3-benzodioxol-5-yl)methylene]hydrazino}-N-(2-hydroxyphenyl)-8-oxooctanamide;8-methoxy-N,N-dimethyl-5H-pyrimido[5,4-b]indol-4-amine;allyl(3R,3′R,4′S,6′R,8′S,8a′S)-6′-{4-[2-({[(3S,6R,7S,8aS)-3-[4-(dimethylamino)butyl]-6-(4-hydroxyphenyl)-1,4-dioxooctahydropyrrolo[1,2-a]pyrazin-7-yl]carbonyl}oxy)ethoxy]phenyl}-5-iodo-1′,2-dioxo-3′,4′-diphenyl-1,2,3′,4′,8′,8a′-hexahydro-1′H-spiro[indole-3,7′-pyrrolo[2,1-c][1,4]oxazine]-8′-carboxylate″bis[4-(dimethylamino)phenyl]methanoneoxime; CN(C)c1ccc2N=c3cc(C)c(N)cc3=Sc2c1;COC(═O)[C@]1(Cc2ccc(O)c(CC═C(C)C)c2)OC(═O)C(═C1c3ccc(O)cc3)O;COc1cc(/C═C/2\Oc3cc(O)ccc3C2=O)ccc1O;COc1cc(ccc1O)c2oc3cc(O)cc(O)c3c(═O)c2O;COc1cc(O)c-2c(CCc3cc(OC)c(OC)cc32)c1;ethyl(2E)-3-(2-hydroxy-5-nitrophenyl)acrylate; ethyl1-benzyl-4-[(dimethylamino)methyl]-5-hydroxy-2-phenyl-1H-indole-3-carboxylate;ethyl 2-ethoxy-5-hydroxy-1H-benzo[g]indole-3-carboxylate; ethyl4-(benzylamino)-6-ethoxyquinoline-3-carboxylate; ethyl4-[({[(5R)-5-ethyl-4,6-dioxo-1,4,5,6-tetrahydropyrimidin-2-yl]thio}acetyl)amino]benzoate;ethyl 4-[(2-phenylethyl)amino]quinoline-3-carboxylate; ethyl4-}[(2-anilino-2-oxoethyl)thio]methyl}-5-hydroxy-2-phenyl-1-benzofuran-3-carboxylate;ethyl 4-{[(2E)-3-(2-thienyl)prop-2-enoyl]amino}benzoate; ethyl6-bromo-4-[(dimethylamino)methyl]-5-hydroxy-1-methyl-2-{[(4-methylphenyl)thio]methyl}-1H-indole-3-carboxylate;ethyl 6-ethoxy-4-{[(1R)-1-methylpropyl]amino}quinoline-3-carboxylate;ethyl 6-methyl-4-[(4-morpholin-4-ylphenyl)amino]quinoline-3-carboxylate;isopropyl(2S)-2-{[(2S)-2-{[(2S,3R)-2-{[(2S)-2-amino-3-mercaptopropyl]amino}-3-methylpentyl]oxy}-3-phenylpropanoyl]amino}-4-(methylsulfonyl)butanoate;methyl(2Z)-2-(4-hydroxybenzylidene)-3-oxo-2,3-dihydro-1-benzofuran-5-carboxylate;methyl 1-hydroxy-3-methylpyrido[1,2-a]benzimidazole-4-carboxylate;methyl2,3-bis-O-(biphenyl-2-ylcarbamoyl)-4-O-[(3-ethylphenyl)carbamoyl]-alpha-L-idopyranoside;methyl5-hydroxy-1-[4-(trifluoromethyl)phenyl]-1H-pyrazole-3-carboxylate;N-(1,3-benzodioxol-5-yl)-7-chloroquinolin-4-amine;N-(2,3-dihydro-1-benzofuran-5-ylcarbonyl)-2-hydroxybenzamide;N-(2,5-dimethylphenyl)benzamide;N-(2-chlorobenzyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;N-(2-chlorobenzyl)-2-phenyl-1H-benzimidazole-5-sulfonamide;N-(2-ethoxyphenyl)-2-hydroxybenzamide;N-(2-hydroxy-4-methylphenyl)-4-[(methylthio)methyl]benzamide;N-(2-hydroxybenzoyl)-2-thiophenecarboxamide;N-(2-hydroxybenzoyl)-3-methoxybenzamide;N-(2-hydroxybenzoyl)-4-(trifluoromethyl)benzamide;N-(2-hydroxyphenyl)-8-[(2E)-2-(1-naphthylmethylene)hydrazino]-8-oxooctanamide;N-(3-bromo-4-hydroxy-1-naphthyl)-4-chlorobenzenesulfonamide;N-(3-chloro-4-hydroxy-1-naphthyl)-4-ethoxybenzenesulfonamide;N-(3-chlorophenyl)-4-(5-hydroxy-1-phenyl-1H-pyrazol-3-yl)piperidine-1-carbothioamide;N-(3-furylcarbonyl)-2-hydroxybenzamide;N-(3-hydroxypyridin-2-yl)-4-phenoxybenzamide;N-(3-imidazo[1,2-a]pyrimidin-2-ylphenyl)cyclopentanecarboxamide;N-(4-bromophenyl)-2-[(3-cyano-4-methyl-6-oxo-1,6-dihydropyridin-2-yl)thio]acetamide;N-(4-carbamoylphenyl)-1-phenyl-3-(2-thienyl)-1H-pyrazole-4-carboxamide;N-(4-ethylbenzoyl)-2-hydroxybenzamide;N-(4-fluorophenyl)-2-(4-hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)acetamide;N-(5-{[(1S)-1-methylpropyl]thio}-1,3,4-thiadiazol-2-yl)-2-(trifluoromethyl)benzamide;N-(5-hydroxy-1-naphthyl)-4-methylbenzenesulfonamide;N-(6-chloro-2-phenyl-4H-chromen-4-ylidene)-1-(2-furyl)methanamine;N-(6-methyl-1,3-benzothiazol-2(3H)-ylidene)thiophene-2-carboxamide;N-(cyclohexylcarbonyl)-2-hydroxybenzamide;N,2-diphenylquinazolin-4-amine;N,N,8-trimethyl-5H-pyrimido[5,4-b]indol-4-amine;N,N′-1H-isoindole-1,3(2H)-diylidenedianiline;N,N-diethyl-8-methyl-5H-pyrimido[5,4-b]indol-4-amine;N,N-dimethyl-4-(6-methylimidazo[1,2-a]pyridin-2-yl)benzenesulfonamide;N-[(1E)-(9-ethyl-9H-carbazol-3-yl)methylene]-4H-1,2,4-triazol-4-amine;N-[(1E)-1H-indol-3-ylmethylene]-1-propyl-1H-benzimidazol-2-amine;N-[(1S)-1-benzylpropyl]-6-[(4-methylpiperidin-1-yl)sulfonyl]-4-oxo-1,4-dihydroquinoline-3-carboxamide;N-[(1S)-1-phenylethyl]quinazolin-4-amine;N-[(3-chloro-1-benzothiophen-2-yl)carbonyl]-2-hydroxybenzamide;N-[(4E)-2-(4-methoxyphenyl)-6-methyl-4H-chromen-4-ylidene]-2-phenylethanamine;N-[2-(1H-benzimidazol-2-yl)phenyl]-2-methylpropanamide;N-[2-chloro-5-(trifluoromethyl)phenyl]-2-(4,4-dimethyl-2,6-dioxocyclohexyl)acetamide;N-[2-hydroxy-3-(4-oxo-4H-chromen-2-yl)phenyl]acetamide;N-[3-(1,3-benzothiazol-2-yl)-4-hydroxyphenyl]-2,2-dimethylpropanamide;N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]benzenesulfonamide;N-[3-(1,3-benzothiazol-2-ylthio)-4-hydroxyphenyl]thiophene-2-sulfonamide;N-[4-(1H-benzimidazol-2-yl)phenyl]-2-(2-methoxyphenyl)acetamide;N-[4-(ethylsulfonyl)-2-hydroxyphenyl]benzamide;N-[5-(ethylsulfonyl)-2-hydroxyphenyl]-2-(4-methoxyphenoxy)acetamide;N4-(3,5-dichlorophenyl)-6-methylpyrimidine-2,4-diamine;N-benzo[g]quinolin-4-yl-N′-isopropylbenzene-1,4-diamine;N-benzyl-2-hydroxybenzamide;N-benzyl-6-{[(3-methoxyphenyl)amino]sulfonyl}-N-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide;N-ethyl-3-phenyl-N-(3-phenylpropyl)propan-1-amine;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c)c(c(O)c2)O[C@@H](Oc3ccc(C(═O)/C═C/c4ccccc4)c(O)c3)[C@@H]1O;O[C@H]1[C@@H](O)[C@@H](COC(═O)c2cc(O)c(O)c(O)c2)O[C@@H](Oc3ccc(C(═O)CCc4ccc(O)cc4)c(O)c3)[C@@H]1O;O[C@H]1[C@@H](Oc2c([C@H]3[C@@H](Oc4cc(O)cc(O)c4C3=O)c5ccc(O)cc5)c(O)cc(O)c2C=O)c6ccc(O)cc6;O[C@H]1[C@H]2[C@H](CC(═O)O)C(═O)O[C@@H]3C(COC(═O)c4cc(O)c(O)c(O)c4)O[C@@H](OC(═O)c5cc(O)c(O)c(O)c5)C(OC(═O)c6cc(O)c(O)c(OC1=O)c26)[C@@H]3OC(═O)c7cc(O)c(O)c(O)c7;OC[C@H]1O[C@@H](OC[C@H]2O[C@@H](Oc3c(oc4cc(O)cc(O)c4c3=O)c5ccc(O)cc5)[C@H](O)[C@@H](O)[C@@H]2O)[C@H](O)[C@@H](O)[C@@H]1O;andOC1[C@H](OC(═O)c2cc(O)c(O)c(O)c2)OC3COC(═O)c4cc(O)c(O)c(O)c4-cc(O)c(O)c(O)cc5C(═O)O[C@@H]1[C@@H]3OC(═O)c6cc(O)c(O)c(O)c6c7c(O)c(O)c(O)cc7C(═O)OOc1ccc(cc1)[C@H]2CC(═O)c3c(O)cc(O)c([C@H]4[C@@H](Oc5cc(O)cc(O)c5C4=O)c6ccc(O)cc6)c3O2Oc1ccc2C(═O)/C(═C/c3ccc(O)c(O)c3)/Oc2c1.